Dengue virus non-structural protein 1 specific binding polypeptides and methods of using the same

ABSTRACT

The present disclosure relates to polypeptides that specifically bind to Dengue virus non¬structural protein 1, including antibodies and fragments thereof. The antibody or antigen-binding fragment thereof may specifically bind Dengue virus (DENV) serotype 4 and include: a heavy chain variable region that comprises at least one CDR amino acid sequence selected from the group consisting of: SGYNWH, YIH YS GGTN YNPS LKS, RTGTVPFAY, SYVMH, YLNPYNDDTKYNEKFKG, and GPPYALDY. The present disclosure further relates to methods of producing the polypeptides of the present disclosure, methods of diagnosing DENV, and methods of treating a DENV infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit and priority to U.S. ProvisionalPatent Application No. 62/353,690, filed on 23 Jun. 2016 and entitledDENGUE VIRUS NON-STRUCTURAL PROTEIN 1 SPECIFIC BINDING POLYPEPTIDES ANDMETHODS OF USING THE SAME, which is incorporated by reference in theirentirety for all purposes.

GOVERNMENT FUNDING

Research supporting this application was carried out by the UnitedStates of America as represented by the Secretary, Department of Healthand Human Services. This research was supported by the IntramuralResearch Program. The Government has certain rights in this invention.

INCORPORATION BY REFERENCE

In compliance with 37 C.F.R. § 1.52(e)(5), the sequence informationcontained in electronic file name: 1420378_441WO2_Sequence_Listing_ST25.txt; size 10.6 KB; created on: 14 May 2017;using Patent-In 3.5, and Checker 4.4.0 is hereby incorporated herein byreference in its entirety.

BACKGROUND 1. Field of the Invention

The present description relates to polypeptides, e.g., antibodies or anantigen-binding fragment thereof, methods of producing the same, andmethods for the treatment and prevention of disease using compositionsof the present disclosure.

2. Background of the Invention

The four dengue virus serotypes (DENV-1 to DENV-4) are extremelyimportant arthropod-borne flaviviruses in terms of morbidity andgeographic distribution. Up to 400 million DENV infections occur everyyear, mostly in tropical and subtropical areas where vector mosquitosare abundant. Infection with any of the DENV may be asymptomatic or maylead to classic dengue fever or more severe dengue hemorrhagic fever(DHF) and dengue shock syndrome (DSS), which are increasingly common inthe dengue endemic areas. Immunity to the same virus serotype (homotypicimmunity) is life-long to that serotype. However, within 2-3 monthsafter infection one is susceptible to other serotype.

Dengue is a serious disease of public importance with increasingworldwide spread. Dengue is caused by infection with any one of the fourantigenically distinct dengue virus serotypes (DENV1-4). At the present,there is no efficacious vaccine and therapeutic agent. Current diagnosisof an acute DENV infection primarily relies on reverse transcriptasepolymerase chain reaction (RT-PCR), a highly sophisticated test. Toimprove dengue case detection in dengue endemic countries a highlysensitive, specific, and simple rapid assay is needed to provide earlysupport for patients and to accurately differentiate dengue from otheracute febrile illnesses. DENV non-structural protein 1 (NS1) is a uniquediagnostic marker for early detection of DENV because it is detected inthe serum of DENV-infected patients as early as one day post onset ofsymptoms (DPO) to 18 DPO at concentrations up to 50 μg/mL.

NS1 is a highly conserved glycoprotein and possesses both group-specificand serotype-specific epitopes hence it has the potential todifferentiate between DENV serotypes.

Rapid tests such as the NS1 enzyme-linked immunosorbent assay (ELISA)are commercially available for DENV with relatively good sensitivity andspecificity. Several recent studies have critically evaluated theperformance of the current commercially available NS1 ELISA kits by theDENV serotypes. However, results from these studies demonstrated thatthese kits are less sensitive for the detection of DENV4. Additionally,these commercial tests had decreased sensitivity in detecting secondarydengue infections, common in dengue endemic countries. Commerciallyavailable NS1 antigen (Ag) tests of Platelia™ (Bio-Rad®, Hercules,Calif., USA) and Dengue Early (Panbio® Diagnostics, Brisbane,Australia), for example, have shown to have low sensitivity to DENV4infections. In addition, Dengue Early test showed 19% sensitivity toDENV4. Furthermore, when comparing the sensitivities between all DENVserotypes, and Platelia™ test had the lowest sensitivity for DENV(58.3%). Collectively, the data establishes that there is a need for anew NS1 Ag detection test/assay with higher sensitivity for DENV4.

In order to improve the performance of NS1 Ag detection tests/assayswith higher sensitivity for DENV4, it is important to understand why thecurrent NS1 Ag detection tests failed to detect DENV4 infectionseffectively. Almost all of the present commercial NS1 Ag detection testsare based on cross-reactive anti-NS1 monoclonal antibodies (MAbs) to allfour DENV serotypes. Several studies have shown the absence ofsignificant amino acid sequence variation in the epitopes ofserotype-cross-reactive MAbs. This is consistent with the observation ofother studies that there was no link between the NS1 gene (amino acid)sequence variation and the poor performance of Dengue Early (Panbio®)for DENV4 detection. Factors other than NS1 gene (and/or amino acid)sequence variation may impact the bio-accessibility and/or binding ofthe conserved epitopes to MAbs depending on the serotype. For example,the bio-accessibility of the conserved epitopes may vary according tothe serotype/genotype when the NS1 protein is folded and assembled toform a NS1 hexamer. It is therefore conceivable to consider that thecommon linear epitopes targeted by MAbs of commercial NS1 tests could beonly partially accessible (i.e., partially inaccessible) on the NS1hexameric isoform of DENV4.

Another factor that could contribute to the poor sensitivity of NS1 Agdetection tests/assays for DENV4 is the low level expression of NS1 inDENV4-infected patients as compared to dengue patients infected with theDENV1, DENV2 and DENV3, although this is not yet properly investigated.Further, most of the anti-NS1 MAbs that have been developed so far andcould be utilized as reagents for development of the current commercialserotype cross-reactive NS1 Ag detection tests were generated fromnative and/or recombinant(r) NS1 of DENV1 and DENV2 immunization. NS1capture ELISAs specific to DENV4 might improve the detection of DENV4cases worldwide. Additionally, the serotype-specific NS1 Ag test canoffer an opportunity to identify DENV serotype. As such, there is a needfor the development of new DENV4 serotype-specific NS1 antibodies andassays that can detect NS1 in the serum of DENV4-infected patients atthe lowest possible detection limit (LOD).

Production of a properly-folded soluble NS1 protein appears to becrucial for the development of MAbs, which are reactive to epitopes thatare accessible on hexameric NS1. However, rNS1 expressed in traditionalexpression system, e.g. Escherichia coli, often results in insolubleaggregates (inclusion bodies). Isolation and purification of proteinsexpressed in this way require solubilization in strong detergents, suchas SDS and urea, which could also lead to denaturation of the targetprotein. Refolding of denatured protein is possible, but attaining thecorrect three-dimensional configuration of the protein is not alwaysachieved or even possible. Expression of rNS1 in Spodoptera frugiperda(Sf) insect cell lines such as Sf9 and Sf21 using a baculovirusexpression system has been utilized as an attractive alternative but, atleast in the inventors' experience (unpublished data), the expressedrNS1 protein was insoluble and required solubilization and refolding,which involves multiple complex steps.

Therefore, a need exists for a DENV4 specific anti-NS1 antibody,especially an antibody directed to epitopes available on hexameric DENV4NS1, and sensitive assays/tests directed to specifically detectingDENV4. In order to circumvent these problems, an expression system thatgenerates soluble and stable rNS1 protein is required.

BRIEF DESCRIPTION

The present disclosure relates to the surprising and unexpecteddiscovery of antibodies that bind specifically to DENV4 NS1 (i.e., bindswith high affinity relative to other DENV4 serotypes), methods ofproducing antibodies directed to multimeric (e.g., hexameric, DENV4NS1), methods of detecting DENV4, and methods of treating and/orpreventing DENV4 infections.

Thus, in certain aspects, the present disclosure provides antibodies andantigen-binding fragments thereof that bind specifically to Dengue virusserotype 4 (DENV4). In certain embodiments, the antibody or antigenbinding fragment thereof comprises: a heavy chain variable region thatcomprises at least one CDR amino acid sequence selected from the groupconsisting of: SGYNWH, YIHYSGGTNYNPSLKS, RTGTVPFAY, SYVMH,YLNPYNDDTKYNEKFKG, and GPPYALDY.

In certain embodiments, the fragment of the antibody is selected fromthe group consisting of a Fab fragment, a F(ab)′, a F(ab)′₂ fragment, ora single-chain variable fragments (scFvs).

In some embodiments, the antibody or fragment thereof is specific forthe DENV Non-structural protein 1 (NS1). In certain embodiments, theantibody or fragment thereof binds specifically to DENV4 NS1.

In other embodiments, the antibody or fragment thereof further comprisesa light chain variable region that includes at least one CDR amino acidsequence selected from the group consisting of: SVSSSISSSNLH, GTSNLAS,QQWSSYPLT, RASQDISNYLN, YTSRLHS, and QQGNTLPRT.

In certain embodiments, the CDR amino acid sequence of the heavy chainvariable region is selected from the group consisting of: SGYNWH,YIHYSGGTNYNPSLKS, and RTGTVPFAY; and the CDR amino acid sequence of thelight chain variable region is selected from the group consisting of:SVSSSISSSNLH, GTSNLAS, and QQWSSYPLT.

In certain other embodiments, the CDR amino acid sequence of the heavychain variable region is selected from the group consisting of: SYVMH,YLNPYNDDTKYNEKFKG, and GPPYALDY; and the CDR amino acid sequence of thelight chain variable region is selected from the group consisting of:RASQDISNYLN, YTSRLHS, and QQGNTLPRT.

In particular embodiments, the heavy chain variable region comprises theamino acid sequence of:

DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYNWHWIRQFPGNKLEWMGYIHYSGGTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCAR RTGTVPFAYWGQGTLVTVSA,or EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYLNPYNDDTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAY GPPYALDYWGQGTSVTVSS.

In particular other embodiments, the light chain variable regioncomprises the amino acid sequence of:

EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLT FGGGTKLEIK, orDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVTLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPRTF GGGTKLEIK.

In yet other embodiment, the heavy chain variable region comprises theamino acid sequence of

DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYNWHWIRQFPGNKLEWMGYIHYSGGTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCAR RTGTVPFAYWGQGTLVTVSA;

and the light chain variable region comprises the amino acid sequence of

EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLT FGGGTKLEIK.

In some embodiments, the heavy chain variable region comprises the aminoacid sequence of

EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYLNPYNDDTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAY GPPYALDYWGQGTSVTVSS;and the light chain variable region comprises the amino acid sequence of

DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVTLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPRTF GGGTKLEIK.

In an embodiment, the antibody is 3H7A9, 6D4B10, or 8A6F2. In anadditional embodiment, the antibody fragment includes an antigen-bindingsite/region from an antibody selected from the group consisting of3H7A9, 6D4B10 or 8A6F2.

In an additional aspect, the description provides a hybridoma thatexpresses an antibody selected from the group consisting of 3H7A9,6D4B10, 8A6F2, or a fragment thereof that includes an antigen-bindingsite/region from an antibody selected from the group consisting of3H7A9, 6D4B10 or 8A6F2, respectively.

An antibody or antigen-binding fragment thereof that binds specificallyto Dengue virus serotype 4 (DENV4), wherein the antibody is: 8A6F2 orthe antigen-binding fragment comprises at least at least one heavy chainor light chain CDR amino acid sequence from an antigen-bindingsite/region of 8A6F2; 3H7A9 or the antigen-binding fragment comprises atleast at least one heavy chain or light chain CDR amino acid sequencefrom an antigen-binding site/region of 3H7A9; or 6D4B10 or theantigen-binding fragment comprises at least at least one heavy chain orlight chain CDR amino acid sequence from an antigen-binding site/regionof 6D4B10.

An additional aspect of the present disclosure provides a pharmaceuticalcomposition. The composition comprises the antibody (or anantigen-binding fragment thereof) of the present disclosure and apharmaceutically acceptable carrier.

Another aspect of the present disclosure provides a method of diagnosingor detecting a DENV4 infection. The method comprises: contacting asample from a patient with the antibody (or an antigen-binding fragmentthereof) of present disclosure; detecting the binding of NS1, whereindetection of DENV4 NS1 is indicative of a patient that is positive forDENV4 infection. In certain embodiments, the antibody is a labeledantibody or detected by a labeled antibody. In certain embodiments, themethod further includes the step of quantifying the binding of theantibody to NS1 of DENV4. In certain embodiments, the method furtherincludes the step of diagnosing the patient as having or not having aDENV4 infection. In certain embodiments the method further includes thestep of administering an effective amount of a treatment effective forameliorating at least one symptom of DENV4 infection.

In some embodiments, contacting the blood sample comprises: contactingthe blood sample to an immobilized antibody (or an antigen-bindingfragment thereof) of the present disclosure; and contacting the antibodyretained DENV4 virion with a second antibody (or an antigen-bindingfragment thereof) of the present disclosure. In an embodiment, theimmobilized antibody (or the fragment thereof) and the second antibody(or the fragment thereof) are different antibodies or fragments thereof.

In other embodiments, the secondary antibody or a fragment thereof islinked to a detectable label. For example, the detectable label may beselected from the group consisting of an enzyme, biotin, streptavidin, aradioactive molecule, and an immunofluorescent protein or dye. Incertain embodiments, when the detectable label is biotin orstreptavidin, the method further comprises contacting a complexcomprising a NS1 and the labeled antibody with a detection molecule thatcomprises streptavidin or biotin, respectively, linked to animmunofluorescent protein or dye, or an enzyme.

In further embodiments, the antibody or fragment thereof is linked to adetectable label and optionally a bead, particle, or nanoparticle. In anembodiment, the bead, particle, or nanoparticle is a magnetic bead.

In certain embodiments, the method further comprises separating acomplex comprising NS1 and the labeled antibody or fragment thereof viathe bead, particle or nanoparticle for detecting the binding of NS1. Inan embodiment, the sample comprises a blood or a tissue sample.

A further aspect of the present disclosure provides a method of treatinga DENV4 infection in a subject. The method comprises: administering to asubject in the need thereof an effective amount of the antibody (or afragment thereof) of the present disclosure or the pharmaceuticalcomposition of the present disclosure, wherein the administering iseffective at treating the infection.

In an embodiment, the antibody (or antigen-binding fragment thereof) isa humanized antibody or antigen-binding fragment thereof.

Additional embodiments relate to a method for preventing or treatingdengue virus infection or a symptom thereof in a mammal includingproviding to the mammal a prophylactically or therapeutically effectiveamount of the composition of any of the embodiments disclosed herein.This method can involve identifying a mammal in need of an agent thatprevents or treats dengue virus infection or a symptom thereof. Theidentification can be by clinical evaluation or evaluation by diagnosticapproach. Some embodiments relate to measuring a marker of dengue virusinfection or a symptom thereof in said mammal. The measurement can be ameasurement of viral load in the mammal.

An additional aspect of the present disclosure provides a method ofproducing/making a DENV NS1 specific antibody or fragment thereof. Themethod comprises: providing a nucleic acid expressing DENV NS1 fusionprotein with a solubility and stability tag; producing a multimeric DENVNS1 complex; and immunizing an animal with the multimeric DENV NS1complex, wherein immunizing the animal produces an antibody specific tothe DENV NS1. For example, the animal may be a chicken, a goat, a guineapig, a hamster, a horse, a mouse, a rat, or a sheep. The multimeric DENVNS1 complex may comprise 2, 3, 4, 5, 6, or more DENV NS1 fusionproteins.

In some embodiments, the method further comprises preparing at least onehybridoma from spleen cells of the immunized animal.

In additional embodiments, the solubility and stability tag includes asecretion signal.

In certain embodiments, the solubility and stability tag is a smallubiquitin-like modifier (SUMO) and/or the secretion signal is gp67.

In other particular embodiments, providing the nucleic acid expressingDENV NS1 fusion protein comprises inserting the Dengue virus NS1 into avector comprising the solubility and stability tag and optionally, thesecretion signal.

In an embodiment, the DENV NS1 is a DENV4 NS1.

In other embodiments, producing the multimeric DENV NS1 complex isperformed with a eukaryotic expression system. In a particularembodiment, the eukaryotic expression system may be a baculovirusexpression system or a vaccinia virus expression system.

In certain embodiments, the producing a multimeric DENV NS1 complex mayinclude a host cell comprising a vector that expresses a serotypespecific DENV NS1 antibody. In another embodiment, the host cell can bea eukaryotic cell, such as a Chinese hamster ovary (CHO) cell, a NS0murine myeloma cell, or a PER.C6® human cell, an insect cell line, Sf9,or Sf21.

In further embodiment, producing a multimeric DENV NS1 complex comprisesinfecting eukaryotic cells with a baculovirus expressing the DENV NS1fusion protein.

In some embodiments, the baculovirus expressing the DENV NS1 fusionprotein is prepared by at least one of: transforming a bacteria with avector comprising the DENV NS1 fusion protein; selecting avector-transformed bacteria; extracting/purifying the vector from thevector-transformed bacteria; transforming a bacteria comprising abaculovirus shuttle vector; selecting a bacteria with a recombinant DENVNS1 fusion protein-baculovirus vector; extracting/purifying therecombinant DENV NS1 fusion protein-baculovirus vector; transfecting aeukaryotic cell with the recombinant DENV NS1 fusion protein-baculovirusvector; or collecting cell culture supernatant comprising thebaculovirus expressing the DENV NS1 fusion protein.

In other embodiments, immunizing the animal with the multimeric DENV NS1complex includes at least one of: administering the multimeric DENV NS1complex to the animal at least two times; isolate at least one primedspleen cell from the animals; fusing the primed spleen cell with amyeloma cell; or selecting a hybridoma cell expressing the antibodyspecific to the DENV NS1.

In a particular embodiment, the method further comprises humanizing theantibody specific for DENV NS1.

In another embodiment, the method further comprises treating theantibody specific for DENV NS1 thereby producing a fragment thereof. Inan embodiment, treating comprises contacting the antibody specific forDENV NS1 with an agent selected from the group consisting of (i) pepsin,(ii) papain, and (iii) pepsin and β-mercaptoethanol.

Other embodiments relate to a vector comprising the nucleic acid of thepeptide of embodiments disclosed herein. Further embodiments relate to ahost cell comprising the vector of embodiments disclosed herein.

Additional embodiments also relate to a cell expressing the antibody (orfragment thereof) of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a partof the specification, illustrate several embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The drawings are only for the purpose ofillustrating an embodiment of the invention and are not to be construedas limiting the invention.

FIG. 1. SUMO*-NS1 fusion gene construct produced in accordance with thepresent disclosure.

FIGS. 2A and 2B. SUMO*-NS1 fusion protein expression analysis inCoomassie-blue stained 12% SDS-PAGE gel is shown in FIG. 2A. Lane 1:SUMO-NS1 under non-reducing conditions; Lane 2: SUMO-NS1 under reducingconditions; and Lane 3: MW of markers in kDa. Western blot ofSUMO*-DENV4 NS1 protein using anti-his antibody is shown in FIG. 2B.Lane1: Molecular Marker; Lane2: secreted NS1; Lane 3: cell lysate ofsolubilized of SUMO*-DENV4 NS1

FIG. 3. Western blot assay to determine the reactivity of hybridomasupernatants to SUMO*-NS1 fusion protein, as well as unfused rNS1protein. M: molecular markers; Lanes 1 and 2: 3H7A9; Lanes 3 and 4:4B6C10; Lanes 5 and 6: 6D4B10; Lanes 7 and 8: 8A6F2; Lanes 9 &10:10H8F7; Lanes 11 and 12: 10H10B5; and Lanes 13 and 14: Antiserum ofmouse ME 1:100, reactivity to SUMO*-DENV NS1 fusion protein and unfusedDENV rNS1, respectively.

FIGS. 4A, 4B, 4C, and 4D. Serotype-specificity of Monoclonal antibodies(MAbs 3H7A9, 8A6F2, 6D4B10 and 10H10B5) for DENV4 as determined byiELISA. The bars show mean optical density at 450 nm, which measures MAbreactivity to NS1s from all four DENV4 serotypes and other flavivirusessuch as Yellow Fever Virus (YFV) and West Nile Virus (WNV) as well asthe SUMO protein that was used as a tag of solubility and stability tothe fusion DENV4 rNS1 protein (FIG. 1). MAbs 3H7A9 (FIG. 4A), 6D4B10(FIG. 4B), and 8A6F2 (FIG. 4C) represent anti-NS1 MAbs specific toDENV4. MAb 10H10B5 (FIG. 4D), which is reactive to SUMO*, represents oneof the three undesirable MAbs generated from immunization of SUMO*-DENV4rNS1 fusion protein, and was included as a control. The OD value forDENV4-serotype MAbs against rNS1 of other flaviviruses and recombinantSUMO protein is below 0. In contrast, MAbs reactive only to SUMOprotein, e.g. 10H10B5, did not show any reactivity to rNS1 of any of theflaviviruses.

FIGS. 5A, 5B, 5C, and 5D. DENV4 serotype-specificity of MAbs 3H7A9,8A6F2, 6D4B10 and 10H10B5 as determined by cell-based ELISA. The barsshow mean optical density at 450 nm that measures MAb's reactivity todimeric NS1 expressed on DENV1-4-infected Vero cells. Mock-infected Verocells were included as a control.

FIGS. 6A and 6B. Analysis of capture/detector MAbs pairs for thedevelopment of DENV4 serotype specific NS1 capture ELISA. The curvesrepresent limit of detection (LOD) curves for matched pairs of MAbs ofthe present disclosure: 6A) 8A6F2/biotinylated 6D4B10, and 6B)6D4B10/biotinylated 8A6F2. The optical density (OD) values at 450 nmwere obtained at various concentration of DENV4 rNS1 with optimalconcentrations for the coating antibody, 10 μg/ml; capture antibody,1:2000 dilution; and streptavidin-tagged horseradish peroxidase(HRP-SP), 1:2000 dilution. West Nile Virus was used as the control.

DETAILED DESCRIPTION

As described herein, the composition or polypeptides of the presentdisclosure bind specifically to DENV4 NS1 (i.e., bind with high affinityrelative to other DENV serotypes), treat and/or prevent a DENV4infection, ameliorate the systems of a DENV4 infection, or anycombination thereof, and therefore, the compositions and/or polypeptidesof the present disclosure represent a novel therapeutic intervention forthe treatment and/or prevention of, for example, DENV4 infections andtissue damage/injury caused therefrom.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. See, e.g., Singleton P andSainsbury D., in Dictionary of Microbiology and Molecular Biology 3rded., J. Wiley & Sons, Chichester, N.Y., 2001. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entireties. In the case of conflict,the present specification, including definitions, will control. Inaddition, the examples are illustrative only and not intended to belimiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise (such as in the case of a groupcontaining a number of amino acids in which case each amino acid numberfalling within the range is provided), between the upper and lower limitof that range and any other stated or intervening value in that statedrange is encompassed within the invention. The upper and lower limits ofthese smaller ranges may independently be included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

The following terms are used to describe the present invention. Ininstances where a term is not specifically defined herein, that term isgiven an art-recognized meaning by those of ordinary skill applying thatterm in context to its use in describing the present invention.

The articles “a” and “an” as used herein and in the appended claims areused herein to refer to one or to more than one (i.e., to at least one)of the grammatical object of the article unless the context clearlyindicates otherwise. By way of example, “an element” means one elementor more than one element.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e., “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.”

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from anyone or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, in certain methods described hereinthat include more than one step or act, the order of the steps or actsof the method is not necessarily limited to the order in which the stepsor acts of the method are recited unless the context indicatesotherwise.

The phrases “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce an adverse,allergic or other undesirable reaction when administered to an animal,or a human, as appropriate.

The terms “co-administration” and “co-administering” or “combinationtherapy” refer to both concurrent administration (administration of twoor more therapeutic agents at the same time) and time variedadministration (administration of one or more therapeutic agents at atime different from that of the administration of an additionaltherapeutic agent or agents), as long as the therapeutic agents arepresent in the patient to some extent, preferably at effective amounts,at the same time. In certain preferred aspects, one or more of thepresent compounds described herein, are co-administered in combinationwith at least one additional bioactive agent, especially including anantiviral, pain reliever, and/or a palliative agent. In particularlypreferred aspects, the co-administration of compounds results insynergistic activity and/or therapy, including antiviral and/or painrelief activity.

The term “amino acid side chain” refers to a moiety attached to theα-carbon in an amino acid. For example, the amino acid side chain foralanine is methyl, the amino acid side chain for phenylalanine isphenylmethyl, the amino acid side chain for cysteine is thiomethyl, theamino acid side chain for tyrosine is 4-hydroxyphenylmethyl, etc. Othernon-naturally occurring amino acid side chains are also included forexample, those that occur in nature (e.g., an amino acid metabolite) orthose that are made synthetically (e.g., an alpha di-substituted aminoacid).

The term polypeptide encompasses two or more naturally occurring orsynthetic amino acids linked by a covalent bond (e.g., an amide bond).Polypeptides as described herein include full length proteins (e.g.,fully processed proteins), as well as shorter amino acids sequences(e.g., fragments of naturally occurring proteins or syntheticpolypeptide fragments).

Throughout the present specification and claims, the numbering of theresidues in an immunoglobulin heavy chain is that of the EU index as inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991). The “EU index as in Kabat” refers to the residue numbering ofthe human IgG1 EU antibody.

The term “homology” is defined as the percentage of residues in amodified amino acid sequence that are identical after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent homology. Methods and computer programs for the alignment arewell known in the art. One such computer program is “ClustalW, which isavailable, for example, on the world-wide web at ebi.ac.uk/clustalw.

An “amino acid modification” refers to a change in the amino acidsequence of a predetermined amino acid sequence. Exemplary modificationsinclude an amino acid substitutions, insertions and/or deletions. Thepreferred amino acid modification herein is a conservative substitution.

An “amino acid substitution” refers to the replacement of at least oneexisting amino acid residue in a predetermined amino acid sequence withanother different “replacement” amino acid residue. The replacementresidue or residues may be “naturally occurring amino acid residues”(i.e., encoded by the genetic code) and selected from the groupconsisting of alanine (Ala); arginine (Arg); asparagine (Asn); asparticacid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu);glycine (Gly); histidine (His); isoleucine (Leu): leucine (Leu); lysine(Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine(Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine(Val). Preferably, the replacement residue is not cysteine. Substitutionwith one or more non-naturally occurring amino acid residues is alsoencompassed by the definition of an amino acid substitution herein. A“non-naturally occurring amino acid residue” refers to a residue, otherthan those naturally occurring amino acid residues listed above, whichis able to covalently bind adjacent amino acid residues(s) in apolypeptide chain. Examples of non-naturally occurring amino acidresidues include norleucine, ornithine, norvaline, homoserine and otheramino acid residue analogues.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

An “amino acid insertion” refers to the incorporation of at least oneamino acid into a predetermined amino acid sequence.

An “amino acid deletion” refers to the removal of at least one aminoacid residue from a predetermined amino acid sequence.

“Hinge region” is generally defined as stretching from Glu216 to Pro230of human IgG1. Hinge regions of other IgG isotypes may be aligned withthe IgG1 sequence by placing the first and last cysteine residuesforming inter-heavy chain S—S bonds in the same positions.

The term “binding domain” refers to the region of a polypeptide thatbinds to another molecule.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multi-specific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity.

“Antibody fragments” (i.e., an antigen-binding fragment of an antibody),as defined for the purpose of the present invention, comprise a portionof an intact antibody, generally including the antigen binding orvariable region of the intact antibody and optionally the Fc region ofan antibody. Examples of antibody fragments include linear antibodies;single-chain antibody molecules (e.g., scFv); F(ab′)₂ fragments; Fab′fragments; and multi-specific antibodies formed from antibody fragments.The antibody fragments may retain at least part of the hinge andoptionally the C_(H)1 region of an IgG heavy chain. The antibodyfragments may retain the entire constant region of an IgG heavy chain,and include an IgG light chain.

The term “Fc region” is used to define a C-terminal region of animmunoglobulin heavy chain. Although the boundaries of the Fc region ofan immunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheFc region of an immunoglobulin generally comprises two constant domains,C_(H)2 and C_(H)3.

The term “F(ab) fragment” is defined as a fragment of an immunoglobulinmolecule that comprises the variable regions of a light chain and aheavy chain. That is, a Fab fragment is a monovalent antigen bindingstructure of an immunoglobulin without the Fc portion, and which resultsfrom the treatment of an immunoglobulin with papain.

The term “F(ab)′ fragment” is defined as a fragment of an immunoglobulinmolecule that comprises the variable regions of a light chain and aheavy chain. That is, the fragment is monovalent and most of the Fcportion is removed, which can be achieved through the treatment of animmunoglobulin molecule with pepsin and β-mercaptoethanol.

The term “F(ab′)₂ fragment” is defined as a fragment of animmunoglobulin molecule that comprises two F(ab) fragments and a portionof the hinge region. That is, most of the Fc portion is removed, whichcan be achieved through the treatment of an immunoglobulin molecule withpepsin.

The term “single-chain variable fragments” (scFvs) is defined as apolypeptide engineered to comprise the variable regions (i.e., theantigen-binding domains) of a light immunoglobulin chain and a heavyimmunoglobulin chain. The light chain and heavy chain can be joined byflexible linker sequence.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein 1975Nature 256:495, or may be made by recombinant DNA methods. The“monoclonal antibodies” may also be isolated from phage antibodylibraries.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody that are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e., residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variabledomain and/or those residues from a “hypervariable loop” (i.e., residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain). “Framework” or “FR” residues are those variable domain residuesother than the hypervariable region residues as herein defined.

An “isolated” polypeptide is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the polypeptide willbe purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornon-reducing conditions using Coomassie blue or, preferably, silverstain. Isolated polypeptide includes the polypeptide in situ withinrecombinant cells since at least one component of the polypeptide'snatural environment will not be present. Ordinarily, however, isolatedpolypeptides will be prepared by at least one purification step.

The term “patient” or “subject” is used throughout the specification todescribe an animal, preferably a human or a domesticated animal, to whomtreatment, including prophylactic treatment, with the compositionsaccording to the present disclosure is provided. For treatment of thoseinfections, conditions or disease states which are specific for aspecific animal such as a human patient, the term patient refers to thatspecific animal, including a domesticated animal such as a dog or cat ora farm animal such as a horse, cow, sheep, etc. In general, in thepresent disclosure, the term patient refers to a human patient unlessotherwise stated or implied from the context of the use of the term.

By “effective amount” is meant the amount required to ameliorate thesymptoms of a disease relative to an untreated patient. The effectiveamount of active compound(s), composition, or component which, when usedwithin the context of its intended use, effects an intended use. Thetherapeutic treatment of a disease varies depending upon the manner ofadministration, the age, body weight, and general health of the subject.Ultimately, the attending physician or veterinarian will decide theappropriate amount and dosage regimen. Such amount is referred to as an“effective” amount. The term effective subsumes all other effectiveamount or effective concentration terms, which are otherwise describedor used in the present application.

As used herein, “treatment”, “treat”, and “treating”, and the likerefers to both therapeutic treatment and prophylactic or preventativemeasures. Those in need of treatment include those already with thedisorder, as well as those in which the disorder is to be prevented. Assuch, these terms refer to reducing or ameliorating a disorder and/orsymptoms associated therewith. It will be appreciated that, although notprecluded, treating a disorder or condition does not require that thedisorder, condition or symptoms associated therewith be completelyeliminated.

As used herein, “ameliorate”, “ameliorating”, or the like refers todecreasing, suppressing, attenuating, diminishing, arresting, orstabilizing the development or progression of a disease.

A “disorder” is any condition that would benefit from treatment with thepolypeptide of the present disclosure. This includes chronic and acutedisorders or diseases including those pathological conditions thatpredispose the mammal to the disorder in question. In one embodiment,the disorder is a viral infection (e.g., a dengue virus disease such asthat which is caused by DENV4).

As used herein, the term “dengue virus disease” means any diseasecaused, directly or indirectly, by one of the four serotypes of a denguevirus, which is a flavivirus. Dengue is an acute febrile diseasecharacterized by sudden onset, with headache, fever, prostration, jointand muscle pain, lymphadenopathy, and a rash that appears simultaneouslywith a temperature rise. A second phase of temperature rise may appearfollowing an afebrile period. Dengue hemorrhagic fever/dengue shocksyndrome is an acute disease occurring primarily in childrencharacterized by an abrupt febrile onset followed by hemorrhagicmanifestations and circulatory collapse.

The word “label” when used herein refers to a detectable compound orcomposition that is conjugated directly or indirectly to thepolypeptide. The label may itself be detectable (e.g., radioisotopelabels or fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or composition thatis detectable.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells. The term “isolated” nucleic acids can mean: (1) amplified invitro by, for example, polymerase chain reaction (PCR); (ii)recombinantly produced by cloning; (iii) purified, as by cleavage andgel separation; or (iv) synthesized by, for example, chemical synthesis.An isolated nucleic acid is one which is readily manipulable byrecombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art.

As used herein with respect to polypeptides, the term “substantiallypure” means that the polypeptides are essentially free of othersubstances with which they may be found in nature or in vivo systems toan extent practical and appropriate for their intended use. Inparticular, the polypeptides are sufficiently pure and are sufficientlyfree from other biological constituents of their host cells so as to beuseful in, for example, generating antibodies, sequencing, or producingpharmaceutical preparations. By techniques well known in the art,substantially pure polypeptides may be produced in light of the nucleicacid and amino acid sequences disclosed herein. Because a substantiallypurified polypeptide of the invention may be admixed with apharmaceutically acceptable carrier in a pharmaceutical preparation, thepolypeptide may comprise only a certain percentage by weight of thepreparation. The polypeptide is nonetheless substantially pure in thatit has been substantially separated from the substances with which itmay be associated in living systems.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmidsand phagemids. A cloning vector is one which is able to replicate in ahost cell, and which is further characterized by one or moreendonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. An expression vector is one into which a desired DNA sequence maybe inserted by restriction and ligation such that it is operably joinedto regulatory sequences and may be expressed as an RNA transcript.Vectors may further contain one or more marker sequences suitable foruse in the identification and selection of cells which have beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., B-galactosidase or alkaline phosphatase), and geneswhich visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques. Preferred vectors are those capable ofautonomous replication and expression of the structural gene productspresent in the DNA segments to which they are operably joined.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants”, “transformed cells”, “transfectants”,and “transfected cells” include the primary subject cell and culturesderived therefrom without regard for the number of transfers. It is alsounderstood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Mutant progeny thathave the same function or biological activity as screened for in theoriginally transformed/transfected cell are included. Where distinctdesignations are intended, it will be clear from the context.

The term “molecular complex” when used herein refers to the relativelystable structure that forms when two or more heterologous molecules(e.g., polypeptides) bind (preferably noncovalently) to one another. Thepreferred molecular complex herein is an immune complex.

“Immune complex” refers to the relatively stable structure that formswhen at least one target molecule and at least one polypeptide of thepresent disclosure bind to one another forming a larger molecular weightcomplex. Examples of immune complexes are antigen-antibody aggregates.

The term “target molecule” refers to a molecule, usually a polypeptide,which is capable of being bound by a heterologous molecule and has oneor more binding sites for the heterologous molecule. The term “bindingsite” refers to a region of a molecule to which another molecule canbind.

Aspects of the present disclosure provide an antibody or anantigen-binding fragment thereof that specifically binds Dengue virusserotype 4 (DENV4), wherein the antibody or an antigen-binding fragmentthereof comprises: a heavy chain variable region that comprises at leastone CDR amino acid sequence selected from the group consisting of:SGYNWH (SEQ ID NO: 1), YIHYSGGTNYNPSLKS (SEQ ID NO: 2), RTGTVPFAY (SEQID NO: 3), SYVMH (SEQ ID NO: 4), YLNPYNDDTKYNEKFKG (SEQ ID NO: 5), andGPPYALDY (SEQ ID NO: 6).

In certain embodiments, the antigen-binding fragment is selected fromthe group consisting of a Fab fragment, a F(ab)′, a F(ab)′₂ fragment, ora single-chain variable fragments (scFvs).

In a particular embodiment, the CDR amino acid sequences of the heavychain variable region are SGYNWH (SEQ ID NO: 1), YIHYSGGTNYNPSLKS (SEQID NO: 2), and RTGTVPFAY (SEQ ID NO: 3). In another particularembodiment, the CDR amino acid sequences of the heavy chain variableregion are SYVMH (SEQ ID NO: 4), YLNPYNDDTKYNEKFKG (SEQ ID NO: 5), andGPPYALDY (SEQ ID NO: 6).

In some embodiments, the antibody or an antigen-binding fragment thereofis specific for the Non-structural protein 1 (NS1).

In other embodiments, the antibody or an antigen-binding fragmentthereof further comprises a light chain variable region that includes atleast one CDR amino acid sequence selected from the group consisting of:SVSSSISSSNLH (SEQ ID NO: 7), GTSNLAS (SEQ ID NO: 8), QQWSSYPLT (SEQ IDNO: 9), RASQDISNYLN (SEQ ID NO: 10), YTSRLHS (SEQ ID NO: 11), andQQGNTLPRT (SEQ ID NO: 12).

In a particular embodiment, the CDR amino acid sequences of the lightchain variable region are SVSSSISSSNLH (SEQ ID NO: 7), GTSNLAS (SEQ IDNO: 8), and QQWSSYPLT (SEQ ID NO: 9). In another particular embodiment,the CDR amino acid sequences of the light chain variable region areRASQDISNYLN (SEQ ID NO: 10), YTSRLHS (SEQ ID NO: 11), and QQGNTLPRT (SEQID NO: 12).

In certain embodiments, the CDR amino acid sequence of the heavy chainvariable region is selected from the group consisting of: SGYNWH (SEQ IDNO: 1), YIHYSGGTNYNPSLKS (SEQ ID NO: 2), and RTGTVPFAY (SEQ ID NO: 3);and the CDR amino acid sequence of the light chain variable region isselected from the group consisting of: SVSSSISSSNLH (SEQ ID NO: 7),GTSNLAS (SEQ ID NO: 8), and QQWSSYPLT (SEQ ID NO: 9). For example, theantibody or an antigen-binding fragment thereof may include the CDRamino acid sequences of the heavy chain variable region are SGYNWH (SEQID NO: 1), YIHYSGGTNYNPSLKS (SEQ ID NO: 2), and RTGTVPFAY (SEQ ID NO:3), and the CDR amino acid sequences of the light chain variable regionare SVSSSISSSNLH (SEQ ID NO: 7), GTSNLAS (SEQ ID NO: 8), and QQWSSYPLT(SEQ ID NO: 9).

In certain other embodiments, the CDR amino acid sequence of the heavychain variable region is selected from the group consisting of: SYVMH(SEQ ID NO: 4), YLNPYNDDTKYNEKFKG (SEQ ID NO: 5), and GPPYALDY (SEQ IDNO: 6); and the CDR amino acid sequence of the light chain variableregion is selected from the group consisting of: RASQDISNYLN (SEQ ID NO:10), YTSRLHS (SEQ ID NO: 11), and QQGNTLPRT (SEQ ID NO: 12). Forexample, the antibody or an antigen-binding fragment thereof may includethe CDR amino acid sequences of the heavy chain variable region areSYVMH (SEQ ID NO: 4), YLNPYNDDTKYNEKFKG (SEQ ID NO: 5), and GPPYALDY(SEQ ID NO: 6), and the CDR amino acid sequences of the light chainvariable region are RASQDISNYLN (SEQ ID NO: 10), YTSRLHS (SEQ ID NO:11), and QQGNTLPRT (SEQ ID NO: 12).

In particular embodiments, the heavy chain variable region comprises theamino acid sequence of:

(SEQ ID NO: 13) DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYNWHWIRQFPGNKLEWMGYIHYSGGTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCAR RTGTVPFAYWGQGTLVTVSA,or (SEQ ID NO: 14) EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYLNPYNDDTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAY GPPYALDYWGQGTSVTVSS.

In particular other embodiments, the light chain variable regioncomprises the amino acid sequence of:

(SEQ ID NO: 15) EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLT FGGGTKLEIK,  or(SEQ ID NO: 16) DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVTLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPRTF GGGTKLEIK.

In yet other embodiment, the heavy chain variable region comprises theamino acid sequence ofDVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYNWHWIRQFPGNKLEWMGYIHYSGGTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARRTGTVPFAYWGQGTLVTVSA (SEQ ID NO:13); and the light chain variable region comprises the amino acidsequence ofEIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGGGTKLEIK (SEQ ID NO: 15).

In some embodiments, the heavy chain variable region comprises the aminoacid sequence ofEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYLNPYNDDTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAYGPPYALDYWGQGTSVTV SS (SEQ IDNO: 14); and the light chain variable region comprises the amino acidsequence of

(SEQ ID NO: 16) DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVTLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPRTF GGGTKLEIK.

In an embodiment, the antibody is 3H7A9, 6D4B10, or 8A6F2. In anadditional embodiment, the antibody fragment includes an antigen-bindingsite/region from an antibody selected from the group consisting of3H7A9, 6D4B10 or 8A6F2.

In an additional aspect, the description provides a hybridoma thatexpresses an antibody selected from the group consisting of 3H7A9,6D4B10, 8A6F2, or a fragment thereof that includes an antigen-bindingsite/region from an antibody selected from the group consisting of3H7A9, 6D4B10 or 8A6F2, respectively.

An antibody or antigen-binding fragment thereof that binds specificallyto Dengue virus serotype 4 (DENV4), wherein the antibody is: 8A6F2 orthe antigen-binding fragment comprises at least at least one heavy chainor light chain CDR amino acid sequence from an antigen-bindingsite/region of 8A6F2; 3H7A9 or the antigen-binding fragment comprises atleast at least one heavy chain or light chain CDR amino acid sequencefrom an antigen-binding site/region of 3H7A9; or 6D4B10 or theantigen-binding fragment comprises at least at least one heavy chain orlight chain CDR amino acid sequence from an antigen-binding site/regionof 6D4B10. For example, the antigen-binding fragment may comprise theheavy chain variable region of 8A6F2 and/or the light chain variableregion of 8A6F2.

An additional aspect of the present disclosure provides a pharmaceuticalcomposition. The composition comprises the antibody (or anantigen-binding fragment thereof) of the present disclosure and apharmaceutically acceptable carrier.

Another aspect of the present disclosure provides a method of diagnosingor detecting a DENV4 infection. The method comprises: contacting asample from a patient with the antibody (or an antigen-binding fragmentthereof) of present disclosure; detecting the binding of NS1, whereindetection of DENV4 NS1 is indicative of a patient that is positive forDENV4 infection. In certain embodiments, the antibody is a labeledantibody or detected by a labeled antibody. In certain embodiments, themethod further includes the step of quantifying the binding of theantibody to NS1 of DENV4. In certain embodiments, the method furtherincludes the step of diagnosing the patient as having or not having aDENV4 infection. In certain embodiments the method further includes thestep of administering an effective amount of a treatment effective forameliorating at least one symptom of DENV4 infection.

In some embodiments, contacting the blood sample comprises: contactingthe blood sample to an immobilized antibody or an antigen-bindingfragment thereof of the present disclosure; and contacting the antibodyretained DENV4 virion with a second antibody or an antigen-bindingfragment thereof of the present disclosure. In an embodiment, theimmobilized antibody (or an antigen-binding fragment thereof) and thesecond antibody (or an antigen-binding fragment thereof) are differentantibodies.

In other embodiments, the secondary antibody (or an antigen-bindingfragment thereof) is linked to (e.g., chemically or covalent linked to)a detectable label. For example, the detectable label may be selectedfrom the group consisting of an enzyme, biotin, streptavidin, aradioactive molecule, and/or an immunofluorescent protein or dye. Incertain embodiments, when the detectable label is biotin orstreptavidin, the method further comprises contacting a complexcomprising a NS1 and the labeled antibody with a detection molecule thatcomprises streptavidin or biotin, respectively, linked to animmunofluorescent protein or dye or an enzyme.

In further embodiments, the antibody (or an antigen-binding fragmentthereof) is linked to (e.g., chemically or covalent linked to) adetectable label and optionally a bead, particle, or nanoparticle. In anembodiment, the bead, particle, or nanoparticle is a magnetic bead.

In certain embodiments, the method further comprises separating acomplex comprising NS1 and the labeled antibody (or an antigen-bindingfragment thereof) via the bead, particle or nanoparticle for detectingthe binding of NS1. In an embodiment, the sample comprises a blood or atissue sample.

A further aspect of the present disclosure provides a method of treatinga DENV4 infection in a subject. The method comprises: administering to asubject in the need thereof an effective amount of the antibody (or anantigen-binding fragment thereof) of the present disclosure or thepharmaceutical composition of the present disclosure, wherein theadministering is effective at treating the infection.

In an embodiment, the antibody (or antigen-binding fragment thereof) isa humanized antibody or antigen-binding fragment thereof.

Other embodiments relate to a vector comprising the nucleic acid of thepeptide of embodiments disclosed herein. Further embodiments relate to ahost cell comprising the vector of embodiments disclosed herein.

Additional embodiments also relate to a cell (e.g., bacterial cells oreukaryotic cells, including hybridomas) expressing the antibody (orfragment thereof) of the present disclosure.

An additional aspect of the present disclosure provides a method ofproducing/making a DENV NS1 specific antibody or fragment thereof. Themethod comprises: providing a nucleic acid expressing DENV NS1 fusionprotein with a solubility and stability tag; producing a multimeric DENVNS1 complex; and immunizing an animal with the multimeric DENV NS1complex, wherein immunizing the animal produces an antibody specific tothe DENV NS1. For example, the animal may be a chicken, a goat, a guineapig, a hamster, a horse, a mouse, a rat, or a sheep. The multimeric DENVNS1 complex may comprise 2, 3, 4, 5, 6, or more DENV NS1 fusionproteins.

In some embodiments, the method further comprises preparing at least onehybridoma from spleen cells of the immunized animal. The hybridomaproduces the DENV NS1 specific antibody.

In other embodiments, the method further comprises expressing the DENVNS1 specific antibody in a host cell. The host cell may comprise anucleic acid that encodes the DENV NS1 specific antibody. The nucleicacid may be operably linked to a transcription regulatory sequence orcontrol sequences. In some additional embodiment, the nucleic acidincludes at least one sequence selected from the group consisting of SEQID NOS: 21-36. In another embodiment, the nucleic acid is comprisedwithin an expression vector.

In additional embodiments, the solubility and stability tag includes asecretion signal.

In certain embodiments, the solubility and stability tag is a smallubiquitin-like modifier (SUMO) and/or the secretion signal is gp67.

In other particular embodiments, providing the nucleic acid expressingDENV NS1 fusion protein comprises inserting the Dengue virus NS1 into avector comprising the solubility and stability tag and optionally, thesecretion signal.

In an embodiment, the DENV NS1 is a DENV4 NS1.

In other embodiments, producing the multimeric DENV NS1 complex isperformed with a eukaryotic expression system. For example, theeukaryotic expression system may be a baculovirus expression system or avaccinia virus expression system. The producing a multimeric DENV NS1complex may include a host cell comprising a vector that expresses aserotype specific DENV NS1 antibody. For example, the host cell can be aeukaryotic cell, such as a Chinese hamster ovary (CHO) cell, a NS0murine myeloma cell, a PER.C6® human cell (LONZA), an insect cell line,Sf9, or Sf21.

In further embodiment, producing a multimeric DENV NS1 complex comprisesinfecting eukaryotic cells with a baculovirus expressing the DENV NS1fusion protein.

In some embodiments, the baculovirus expressing the DENV NS1 fusionprotein is prepared by at least one of: transforming a bacteria (e.g.,DH5α™ E. coli) with a vector (e.g., pI-secSUMO*) comprising the DENV NS1fusion protein; selecting a vector-transformed bacteria;extracting/purifying the vector from the vector-transformed bacteria;transforming a bacteria comprising a baculovirus shuttle vector (e.g.,DH10bac™ E. coli); selecting a bacteria with a recombinant DENV NS1fusion protein-baculovirus vector; extracting/purifying the recombinantDENV NS1 fusion protein-baculovirus vector; transfecting a eukaryoticcell (e g a mammalian cell, or insect cell, e.g., Sf9 or Sf21) with therecombinant DENV NS1 fusion protein-baculovirus vector; or collectingcell culture supernatant comprising the baculovirus expressing the DENVNS1 fusion protein.

In other embodiments, immunizing the animal with the multimeric DENV NS1complex includes at least one of: administering the multimeric DENV NS1complex to the animal at least two times (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 administrations); isolate at least one primed spleen cell fromthe animals; fusing the primed spleen cell with a myeloma cell (e.g., amouse myeloma cell or a rat myeloma cell); or selecting a hybridoma cellexpressing the antibody specific to the DENV NS1.

In a particular embodiment, the method further comprises humanizing theantibody specific for DENV NS1.

In another embodiment, the method further comprises treating theantibody specific for DENV NS1 thereby producing a fragment thereof. Inan embodiment, treating comprises contacting the antibody specific forDENV NS1 with an agent selected from the group consisting of (i) pepsin,(ii) papain, and (iii) pepsin and β-mercaptoethanol.

Other embodiments relate to a vector comprising the nucleic acid of thepolypeptide of the present disclosure. Further embodiments relate to ahost cell comprising the vector of the present disclosure. For example,the nucleic acid of the polypeptide (which can be included in a in ahost cell for expression) can include a heavy chain variable regionsequence selected from the group consisting of:

(SEQ ID NO: 21) GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGGTTATAACTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATACACTACAGTGGTGGCACTAACTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGAAGGACTGGGACGGTCCCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCA CTGTCTCTGCA and(SEQ ID NO: 22) GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATGTTATGCACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATCTTAATCCTTACAATGATGATACTAAGTACAATGAGAAGTTCAAAGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCCTACGGCCCTCCCTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCG TCTCCTCA,which results in amino acid sequences SEQ ID NOS: 13 and 14.

In other embodiments, the nucleic acid of the polypeptide can include alight chain variable region sequence selected from the group consistingof

(SEQ ID NO: 23) GAAATTGTGCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGAGAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCAACTTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTATGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTCTCACAATCAGCAGTATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTGGAGTAGTTACCCACTCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA and (SEQ ID NO: 24)GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTACACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA,which results in amino acid sequences SEQ ID NOS: 15 and 16.

In another embodiment, the nucleic acid of the polypeptide includes aheavy chain variable region selected from the group consisting ofAGTGGTTATAACTGGCAC (SEQ ID NO: 25),TACATACACTACAGTGGTGGCACTAACTACAACCCATCTCTCAAAAGT (SEQ ID NO: 26),AGGACTGGGACGGTCCCGTTTGCTTAC (SEQ ID NO: 27), GGCCCTCCCTATGCTTTGGACTAC(SEQ ID NO: 28), TATCTTAATCCTTACAATGATGATACTAAGTACAATGAGAAGTTCAAAGGC(SEQ ID NO: 29), and AGCTATGTTATGCAC (SEQ ID NO: 30). In otherembodiments, the nucleic acid of the polypeptide includes a light chainvariable region selected from the group consisting of

(SEQ ID NO: 31) AGTGTCAGCTCAAGTATAAGTTCCAGCAACTTGCAC, (SEQ ID NO: 32)GGCACATCCAACCTGGCTTCT, (SEQ ID NO: 33) CAACAGTGGAGTAGTTACCCACTCACG,(SEQ ID NO: 34) AGGGCAAGTCAGGACATTAGCAATTATTTAAAC, (SEQ ID NO: 35)CAACAGGGTAATACGCTTCCTCGGACG, and (SEQ ID NO: 36) TACACATCAAGATTACACTCA.

Additional embodiments also relate to a cell (e.g., bacterial cells oreukaryotic cells, including hybridomas) expressing the antibody (orfragment thereof) of the present disclosure.

Production of Antibodies

A description follows as to exemplary techniques for the production ofthe antibodies used in accordance with embodiments disclosed herein.

(i) Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(ii) Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler and Milstein 1975 Nature 256:495, ormay be made by recombinant DNA methods.

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59 103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are SP-2, SP 2/0, or X63-Ag8-653 cellsavailable from the American Type Culture Collection, Manassas, Va. USA.Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Brodeur etal., Monoclonal Antibody Production Techniques and Applications, pp. 5163 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). The binding affinity of the monoclonalantibody can, for example, be determined by the Scatchard analysis ofMunson et al., Anal. Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce antibody protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy chain and light chain constant domains in placeof the homologous murine sequences, or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

(iii) Humanized Antibodies

Methods for humanizing non-human antibodies have been described in theart. Preferably, a humanized antibody has one or more amino acidresidues introduced into it from a source that is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522 525 (1986)), bysubstituting hypervariable region sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some hypervariable region residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence that is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody. Another methoduses a particular framework region derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies.

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablethat illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

The present application also contemplates affinity matured antibodies,which antibodies bind antigen. The parent antibody may be a humanantibody or a humanized antibody. The affinity matured antibodypreferably binds to antigen with an affinity superior to that of theparent antibody.

Various forms of the humanized or affinity matured antibody arecontemplated. For example, the humanized or affinity matured antibodymay be an antibody fragment. Alternatively, the humanized or affinitymatured antibody may be an intact antibody, such as an intact IgG1 orIgG2b antibody.

(iv) Human Antibodies

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.

As discussed above, human antibodies may also be generated by in vitroactivated B cells.

(v) Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies. However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove.

(vi) Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Methods for making bispecificantibodies are known in the art. Traditional production of full lengthbispecific antibodies is based on the coexpression of two immunoglobulinheavy chain-light chain pairs, where the two chains have differentspecificities. Because of the random assortment of immunoglobulin heavyand light chains, these hybridomas (quadromas) produce a potentialmixture of 10 different antibody molecules, of which only one has thecorrect bispecific structure. Purification of the correct molecule,which is usually done by affinity chromatography steps, is rathercumbersome, and the product yields are low.

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, C_(H)2, and C_(H)3 regions. It is preferred tohave the first heavy-chain constant region (C_(H)1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation.

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimersthat are recovered from recombinant cell culture. The preferredinterface comprises at least a part of the C_(H)3 domain of an antibodyconstant domain. In this method, one or more small amino acid sidechains from the interface of the first antibody molecule are replacedwith larger side chains (e.g., tyrosine or tryptophan). Compensatory“cavities” of identical or similar size to the large side chain(s) arecreated on the interface of the second antibody molecule by replacinglarge amino acid side chains with smaller ones (e.g., alanine orthreonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells.Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents are well known inthe art, along with a number of cross-linking techniques.

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared.

(vii) Other Amino Acid Sequence Modifications

Amino acid sequence modification(s) of the antibodies described hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody.Amino acid sequence variants of the antibody are prepared by introducingappropriate nucleotide changes into the antibody nucleic acid, or bypeptide synthesis. Such modifications include, for example, deletionsfrom, and/or insertions into and/or substitutions of, residues withinthe amino acid sequences of the antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe antibody, such as changing the number or position of glycosylationsites.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis”. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody a polypeptide that increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin Table 1 under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table 1,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE 1 Amino Acid Substitutions Original Residue ExemplarySubstitutions Preferred Substitutions Ala (A) Val, Leu, Ile Val Arg (R)Lys, Gln, Asn Lys Asn (N) Gln, His, Asp, Lys, Arg Gln Asp (D) Glu, AsnGlu Cys (C) Ser, Ala Ser Gln (Q) Asn, Glu Asn Glu (E) Asp, Gln Asp Gly(G) Ala Ala His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala,Phe, Leu norleucine Leu (L) Norleucine, Ile, Val, Met, Ile Ala, Phe Lys(K) Arg, Gln, Asn Arg Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile,Ala, Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W)Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe,Ala, Leu norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

hydrophobic: norleucine, met, ala, val, leu, ile;

neutral hydrophilic: cys, ser, thr;

acidic: asp, glu;

basic: asn, gln, his, lys, arg;

residues that influence chain orientation: gly, pro; and

aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

To increase the serum half-life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody. As used herein, theterm “salvage receptor binding epitope” refers to an epitope of the Fcregion of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that isresponsible for increasing the in vivo serum half-life of the IgGmolecule. The next section describes approaches to design and generateimmunoadhesins.

Pharmaceutical Formulations

Prophylactic or therapeutic formulations of the antibodies used inaccordance with embodiments disclosed herein are prepared for storage bymixing an antibody having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences, 18^(th) ed., 1990, Mack PublishingCo., Easton, Pa.), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorchinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being prevented or treated,preferably those with complementary activities that do not adverselyaffect each other. For example, it may be desirable to further provideantibodies that bind to other targets (e.g., an antibody that binds adifferent epitope). Alternatively, or additionally, the composition mayfurther comprise antiviral agent(s) or pain reliever(s), such asanalgesics. Such molecules are suitably present in combination inamounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin micropheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 18th ed., 1990, Mack PublishingCo., Easton, Pa.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides,copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable micropheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. The formulations to be used for invivo administration must be sterile. This is readily accomplished byfiltration through sterile filtration membranes. The following sectiondescribes alternative, non-therapeutic uses for the polypeptide ofembodiments disclosed herein.

Non-Therapeutic Uses for the Polypeptide

The polypeptide may be useful in diagnostic assays, e.g., for detectingexpression of an antigen of interest in specific cells, tissues, orserum. For diagnostic applications, the polypeptide typically will belabeled with a detectable moiety. Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I,³H, and ¹³¹I are available. The polypeptide can be labeled with theradioisotope using the techniques described in Current Protocols inImmunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, NewYork, N.Y., 1991, for example, and radioactivity can be measured usingscintillation counting. Fluorescent labels such as rare earth chelates(europium chelates) or fluorescein and its derivatives, rhodamine andits derivatives, dansyl, Lissamine, phycoerythrin and Texas Red areavailable. The fluorescent labels can be conjugated to the polypeptideusing the techniques disclosed in Current Protocols in Immunology,supra, for example. Fluorescence can be quantified using a fluorimeter.In addition, various enzyme-substrate labels are available. The enzymegenerally catalyzes a chemical alteration of the chromogenic substratethat can be measured using various techniques. For example, the enzymemay catalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. The chemiluminescentsubstrate becomes electronically excited by a chemical reaction and maythen emit light that can be measured (using a chemiluminometer, forexample) or donates energy to a fluorescent acceptor. Examples ofenzymatic labels include luciferases (e.g., firefly luciferase andbacterial luciferase, luciferin, 2,3-dihydrophthalazinediones, malatedehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO),alkaline phosphatase, [ß-galactosidase, glucoamylase, lysozyme,saccharide oxidases (e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase), heterocyclic oxidases (such asuricase and xanthine oxidase), lactoperoxidase, microperoxidase, and thelike.

Sometimes, the label is indirectly conjugated with the polypeptide. Theskilled artisan will be aware of various techniques for achieving this.For example, the polypeptide can be conjugated with biotin and any ofthe three broad categories of labels mentioned above can be conjugatedwith avidin, or vice versa. Biotin binds selectively to avidin and thus,the label can be conjugated with the polypeptide in this indirectmanner. Alternatively, to achieve indirect conjugation of the label withthe polypeptide, the polypeptide is conjugated with a small hapten(e.g., digoxigenin) and one of the different types of labels mentionedabove is conjugated with an anti-hapten polypeptide (e.g.,anti-digoxigenin antibody). Thus, indirect conjugation of the label withthe polypeptide can be achieved. In another embodiment, the polypeptideneed not be labeled, and the presence thereof can be detected using alabeled antibody that binds to the polypeptide.

The polypeptide of embodiments disclosed herein may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays (e.g., ELISA), immunoprecipitation assays,immunohistochemistry, and/or flow cytometry. The polypeptide may also beused for in vivo diagnostic assays. Generally, the polypeptide islabeled with a radionuclide (such as ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H,³²P or ³⁵S) so that the antigen or cells expressing it can be localizedusing immunoscintiography.

In Vivo Uses for the Polypeptide

It is contemplated that the polypeptide of embodiments disclosed hereinmay be used for the prophylaxis or treatment of a mammal, e.g. a patientsuffering from, or predisposed to, a disease or disorder that couldbenefit from administration of the polypeptide of the presentdisclosure.

The polypeptide of the present disclosure can be administered by anysuitable means, including parenteral, subcutaneous, intraperitoneal,intrapulmonary, and intranasal. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the polypeptide is suitablyadministered by pulse infusion, particularly with declining doses of thepolypeptide. Preferably the dosing is given by injections, mostpreferably intravenous or subcutaneous injections, depending in part onwhether the administration is brief or chronic.

For the prevention or treatment of disease, the appropriate dosage ofthe polypeptide of the present disclosure will depend on the type ofdisease to be prevented or treated, the severity and course of thedisease, whether the polypeptide of the present disclosure isadministered for preventive or therapeutic purposes, previousprophylaxis and therapy, the patient's clinical history and response tothe polypeptide of the present disclosure, and the discretion of theattending physician. The polypeptide of the present disclosure issuitably administered to the patient at one time or over a series oftreatments.

For passive immunization with an antibody, about 1 μg/kg to 15 mg/kg(e.g., 0.1 20 mg/kg) of the polypeptide of the present disclosure is aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the prophylaxis or treatment is sustained until a desired suppression ormodification of disease symptoms occurs. However, other dosage regimensmay be useful. The progress of this therapy is easily monitored byconventional techniques and assays.

The composition comprising a polypeptide of the present disclosure willbe formulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being prevented or treated, the particular mammalbeing treated, the clinical condition of the individual patient, thecause of the disorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The “prophylactically or therapeuticallyeffective amount” of the polypeptide to be administered will be governedby such considerations, and is the minimum amount necessary to prevent,ameliorate, or treat a disease or disorder. The polypeptide of thepresent disclosure need not be, but is optionally formulated with one ormore agents currently used to prevent or treat the disease in question.The effective amount of such other agents depends on the amount ofpolypeptide present in the formulation, the type of disorder ortreatment, and other factors discussed above. These are generally usedin the same dosages and with administration routes as used hereinbeforeor about from 1 to 99% of the heretofore employed dosages.

The following examples provide illustrations of some of the embodimentsdescribed herein but are not intended to limit invention.

Examples

Materials and Methods of the Examples:

Preparation of NS1 Antigen from DENV-Infected Vero Cells. DENV infectedVero cell culture supernatant was produced from each of the four DENVserotypes was produced as previously described (Gelanew, Poole-Smith,and Hunsperger 2015) with a slight modification. Briefly, Vero cells(ATCC®, Manassas, Va., USA) were infected at a multiplicity of infection(MOI)=0.1 and grown in M199 medium (Gibco®/Life Technologies, GrandIsland, N.Y., USA) supplemented with 5% super low IgG FBS (Invitrogen™Carlsbad, Calif., USA) and 1% gentamicin (Gibco®, Grand Island, N.Y.,USA), 1% penicillin-streptomycin (Gibco®, Grand Island, N.Y., USA), 3%sodium bicarbonate (Gibco®, Grand Island, N.Y., USA). In order to avoidcell lysis, and thereby the release of proteolytic enzymes into theculture medium, DENV-infected Vero cells were monitored every day forcytopathic effect (CPE), and culture supernatants were harvested andfiltered on day 3 or 4 post infection at approximately 50% CPE. The sameprocedure was followed for control antigen from mock-infected Verocells. Culture medium was concentrated (about 3×) by using Amicon®,Centricon®-30K (Millipore, Billerica, Mass., USA) and treated withSIGMAFAST™ protease inhibitor cocktail (Sigma-Aldrich®, St. Louis, Mo.,USA). The presence of secreted native NS1 in these culture supernatantswas confirmed using a serotype-cross-reactive Dengue Early NS1 ELISA(Panbio® Diagnostics, Brisbane, Australia). Thereafter, these culturesupernatants were stored at ⁻80° C. until use.

Cloning DENV4 NS1 into pI-secSUMO* Vector. Total viral RNA was isolatedfrom viral seeds of DENV4 (H421, a prototype strain) infected C6/36cells (Aedes albopictus cell line, ATCC®) using the QIAamp® Viral RNAMini kit (QIAGEN®, Gaithersburg, Md., USA), and a complimentary DNA(cDNA) of DENV4 NS1 gene was reverse transcribed using Super Script™ IIIone step reverse transcriptase polymerase chain reaction (RT PCR) kit(Invitrogen™, Carlsbad, Calif., USA) and paired primers

(forward: 5′-ATACGTCTCTAGGTGACACGGGTTGTGCGGTG-3′; SEQ ID NO: 17) and(reverse; 5′-GCGTCTAGATTAGGCCGATACCTGTGATTT-3; SEQ ID NO: 18),according to manufacturer's instructions. These primers were designed tointroduce restriction sites for BmsB1 and Xbal in order to clone thefull length of DENV4 NS1 gene into a directional pI-secSUMO* cloningvector (LifeSensors, Malvern, Pa.). Prior to primer design, the completeabsence of BmsB1 and Xbal restriction sites in the full-length of DENV4NS1 gene sequence was confirmed using a NEBcutter V2.0(nc2.neb.com/NEBcutter2/).

The underlined nucleotide sequences in the forward and reverse primersrepresent the actual first codons, and the complementary sequence to thelast codons of the DENV4 NS1 gene, respectively. At the 5′ end of theforward primer, ATA was added to enhance the efficacy of BmsB1digestions of the PCR product. The sequence CGTCTCT was added as a BmsB1restriction cleavage site for the insertion of NS1 into the multiplecloning sites of the pI-secSUMO* vector, and GGT represented the lastcodon of the SUMO* tag. In the 5′ end of the reverse primer, GCG wasadded to enhance the efficacy of Xbal restriction digest of the PCRproduct end, TCTAGA was added to serve as an Xbal restriction cleavagesite for the insertion of NS1 into the multiple cloning site of thepI-secSUMO* vector, and TTA represented a reverse complement of a stopcodon.

The generated amplicons were visualized by gel electrophoresis with 1%low melting point agarose (LMPA; Invitrogen™, Calsbad, Calif., USA), andthe appropriated band was excised, and purified using a QIAquick® GelExtraction Kit (QIAGEN®, Gaithersburg, Md., USA) and double restrictionenzyme digested with BmsB1 and Xbal (New England BioLabs®, Ipswich,Mass., USA). Since the optimal temperatures and buffers for these tworestriction enzymes are different, a two-step restrictions digest wasperformed. First, amplicons were digested with Xbal at 37° C. for 1hour, the solution was then centrifuged at 10,000×g for 30 minutes, andthe supernatant discarded. The DNA pellet obtained was washed twice withan ice cold 70% ethanol, air dried, and re-suspended in Milli-Q® water.The re-suspended DNA pellet was digested with BsmB1 for 1 hour at 55° C.Then pI-secSUMO* plasmids were linearized with BsmB1 in order togenerate two overhang ends which are complementary to thedouble-digested PCR product ends. Both the double-digested PCR productsand linearized plasmids were then visualized on a 2% LMPA gel. Theappropriate bands were excised, purified as described above, and ligatedovernight at 16° C. using T4 DNA Ligase (Invitrogen™, Calsbad, Calif.,USA) with a 3:1 ratio of double-restricted amplicons (NS1 gene) tolinearized pI-secSUMO* plasmids. The ligated solution was thenimmediately transformed into chemically competent DH5a E. coli cells(Invitrogen™, Calsbad, Calif., USA) per the transformation protocoldescribed in Panavas et al. (2009). Positive DH5a E. coli clones withrecombinant SUMO*-NS1 gene were then selected by colony PCR usingforward primer polH (described below) and the reverse primer describedabove. Additionally, to verify the integrity of the NS1 gene,recombinant plasmid DNAs isolated from PCR-positive clones usingQIAprep® Skin Miniprep Kit (QIAGEN®, Gaithersburg, Md., USA) weresequenced in both directions using two external primers (polH,5′-GGATTATTCATACCGTCCCACCAT-3′ (SEQ ID NO: 19) and Tn7, 5′-CTGGGTGTAGCGTCGTAAGCTAATAC-3′ (SEQ ID NO: 20)). Plasmid DNAs with SUMO*-NS1fusion constructs were then transformed into chemically competentDH10bac E. coli cells (Invitrogen™, Calsbad, Calif., USA) according tothe transformation protocol described in Panavas et al. (2009). Positiveclones were selected by blue and white screening assay followed bycolony PCR. Then Bacmid DNA was isolated from positive clones usingQIAprep® Spin Miniprep Kit (QIAGEN®, Gaithersburg, Md., USA), andsequenced for integrity in both direction using primer pair polH andTn7. The isolated Bacmid DNAs were stored at 4° C. until use.

Expression of SUMO*-DENV4 NS1 Fusion Protein in Sf21 Cells. SUMO*-NS1fusion protein was expressed in Spodoptera frugiperda (Sf)21 cells(Invitrogen™, Calsbad, Calif., USA). Briefly, Sf21 cells weretransfected using 3 μg of bacmid DNA for high titer viral stockproduction, which was later used to infect of these cells at an MOI=1.Because pI-secSUMO* plasmid contain an upstream gp67 secretion signal ofthe SUMO* fusion which could result in secretion of the SUMO*-rNS1 intothe cell culture medium (FIG. 1), only culture supernatant of Sf21 cellswas harvested on day 3 post infection. The culture supernatants(containing soluble SUMO*-NS1 fusion protein) was concentrated (5×) andthen purified by immobilized metal affinity chromatography (IMAC) undernative conditions. The purity, as well as the molecular size, of thefusion protein was determined by Coomassie blue stained 12% SDS-PAGE gel(Invitrogen™, Calsbad, Calif., USA) under both reducing and non-reducingconditions, and confirmed by western blot assay. Proper folding of theSUMO* tagged DENV-4 rNS1 protein was confirmed by commercial serotypecross-reactive Dengue Early (Panbio® Diagnostics, Brisbane, Australia),which comprises MAbs reactive to the native hexameric NS1 in serumsamples of dengue patients.

Western blot analysis. Fused protein of SUMO*-DENV-4 NS1 and unfusedDENV4 rNS1 protein at concentrations of 2 μg or 100 ng were separatedusing NuPAGE® Novex™12% SDS-PAGE gel (Life Technologies™ Corporation,Carlsbad, Calif., USA) under non-reduced, heat-denatured ornon-denatured conditions. Protein size discrimination was determinedwith Molecular Weight (MW) Standards (116, 66, 45, 31, and 21 kDa) andMagicMark™ XP Protein standards (20-220 kDa, Invitrogen™, Calsbad,Calif., USA). For western blot analysis the proteins were transferred to0.22 μm nitrocellulose membranes (Invitrogen™, Calsbad, Calif., USA),blocked overnight at room temperature (RT) in 5% NFDM in phosphatebuffered saline (PBS) with 0.05% Tween20 (NFDM-PBST). For SUMO*-DENV-4NS1 protein analysis by western blot, the membrane was incubated withanti-HisTag. For the characterization of MAbs elicited forSUMO*-DENC4-NS1, the nitrocellulose membranes were incubated with theprimary MAbs that were produced against the fusion protein. Followingthe incubation with primary antibody (i.e., the generated MAbs), asecondary anti-mouse peroxidase detector antibody diluted at 1:10,000(KPL, Gaithersburg, Md., USA) was used. The substrate SuperSignal™ WestPico solution (Pierce™/Thermo Scientific™, Rockford, Ill., USA) was usedto detect proteins.

Mice Immunization and Isolation of Anti-DENV4 NS1 MAbs SecretingHybridoma Clones. The immunization and production of hybridoma cloneswere performed by Custom Antibody Generation Services (CelteinBiosciences, LLC; Monroe, Ohio, USA) using our SUMO*-DENV4 NS1 fusionprotein. To produce anti-NS1 MAb-secreting hybridoma clones, femaleBALB/c mice (6-8 weeks) were immunized with purified soluble SUMO*-DENV4NS1 fusion protein in complete Freud's adjuvant followed by four boostswith incomplete Freund's adjuvant with 14 day intervals between boosts.Four days after the last boost, primed spleen cells were isolatedaseptically from mice with the highest titer against SUMO*-DENV4 NS1fusion protein and unfused DENV4 rNS1 (expressed in mammalian cells).These primed spleen cells were then fused with SP2/0 cells as previouslydescribed (Kohler and Milstein, 1975). To select desired MAb secretinghybridomas, culture supernatant from each clone was evaluated by iELISAusing both SUMO*-DENV4 NS1 fusion protein and unfused DEN4 rNS1 proteinand fixed cell based ELISA as described previously in Gelanew et al.(2015). Further, the hybridoma culture supernatants were also screenedagainst recombinant SUMO protein by iELISA.

MAb Production, Purification, and Characterization. MAb production frompositive hybridoma clones (3H7A9, 6D4B10, and 8A6F2) was carried outusing a CELLine™ 1000 System bioreactor (BD™ Biosciences; Sparks, Md.,USA) according to the manufacturer's instructions and as per protocolsdescribed in Gelanew et al. (2015). Purification MAb was done usingVivapure® Maxiprep Protein G Spin Columns (Satorius™ Stedim, Bohemia,N.Y., USA) per the manufacturer's instructions. MAbs were characterizedusing fixed cell ELISA and iELISA as previously described (Gelanew,Poole-Smith, and Hunsperger 2015).

MAbs isotypes were determined using a Mouse MAbs Isotyping kit(Pierce™/Thermo Scientific™, Rockford, Ill., USA) according to themanufacturer's instructions. Purified and dialyzed MAbs werebiotinylated with a spacer arm biotin (NHS-PEG₄-Biotin) using EZ-Link™NHS-PEG4-Biotinylation Kit (Pierce™/Thermo Scientific™, Rockford, Ill.,USA).

Epitope Mapping by Competition ELISA. In order to determine whether theMAbs recognize the same epitope or distinct epitopes on the DENV4 NS1, acompetition ELISA was performed as described in Gelanew et al. (2015).

Sequencing the Antigen Binding Sites of MAbs. Sequencing the antigenbinding sites (the variable regions of light (V_(L)) and heavy (V_(H))chains) of the three MAbs were performed by customer service (GenScript,N.J., USA) in order to determine the identity of the MAbs. Five singlecolonies with the correct variable light chain and heavy chain genes ofeach hybridoma clones was sequenced. After performing a multiplesequence alignment of the five peptide sequences using ClustalW2(www.ebi.ac.uk), a consensus sequence for both the V_(L) and the V_(H)of each hybridoma clone was obtained. Finally, multiple alignments wereperformed among V_(L) consensus sequences and V_(H) consensus sequencesto determine the identity of binding region of the three complementaritydetermining regions (CDR1, CDR2, and CDR3) of MAbs.

Development of DENV4 Specific NS1 Capture ELISA. NS1 capture ELISAs weredeveloped by utilizing two of the best MAbs as a capture and a detectorantibodies. In order to determine the optimal capture/detector pair forthe detection of DENV4 NS1 antigen, each MAbs was tested either as acapture or a detector antibody. The optimal concentrations of captureand detector antibodies were determined by checkerboard titration ELISA.Microtiter plates were coated with MAb 8A6F2 (100 μl/well) diluted inbicarbonate buffer (pH 9.6) at a concentration of 10 μg/mL and incubatedovernight at 4° C. The next day, excess unbound capture MAb was removed,and thereafter, the microtiter plates were blocked with 200 μl/well of5% NFDM or 2% bovine serum albumin (BSA) (w/v). After a 45 minuteincubation, the microtiter plates were washed 3× with PBST. Thereafter,100 μl/ml of rNS1 antigen (0.5 μg/ml) and serum sample diluted 1:1 inPBST was added and incubated at 37° C. for 60 minutes. Following theincubation and three washes with PBST, 100 μl/well of biotinylated MAb6D4B10 diluted at 1:2000 in PBST was added and incubated for 1 hour at37° C. After 3 washes with PBST, 100 μl/well of peroxidase-conjugatedstreptavidin was added. The microtiter plates were then incubated at 37°C. for 30-60 minutes. After 5 washes with PBST, the reaction wasvisualized by adding 100 μl/well of 3,3′,5,5′-Tetramethylbenzidine (TMB)liquid substrate and incubating at RT in the dark for 15 minutes. Thereaction was then stopped with 100 μl/well of TBM stop solution. Theoptical density (OD) was measured at 450 nm on ELISA ELx800™ microplatereader (BioTek®, Vermont, USA). Each sample was tested in triplicate.

Testing Culture Supernatants from DENV1-4 Infected Vero Cells and rNS1of Flaviviruses. The serotype-specificity of the above described NS1capture ELISA was examined using culture supernatants obtained fromDENV-infected Vero cells and commercially available rNS1 ofFlaviviruses, including all four DENV serotypes and expressed inmammalian cell line (NativeAntigen, Oxfordshire, UK). The presence ofNS1 in the culture supernatants was pre-confirmed using Dengue Early(Panbio® Diagnostics, Brisbane, Australia).

SUMO*-DENV4 NS1 Fusion Protein Expressed was the Correct Conformation ofDENV NS1. Affinity purified SUMO*-DENV4 NS1 fusion protein expressed inSf21 cells appeared as a monomer (approximately 62 kDa) and a dimer(approximately 120 kDa) on reduced and non-reduced 12% SDS-PAGE,respectively (FIG. 2A). The expected sizes of monomeric and dimericnative DENV4 NS1 expressed in mammalian cells are 48 kDa and 80 kDa,respectively (Flamand et al. 1999). Considering the molecular weight ofthe SUMO* tag, which is about 11.5 kDa, the observed sizes on SDS-PAGEmatched the expected sizes of monomeric and dimeric DENV4 NS1,respectively. Also, the appearance of a single band on SDS-PAGE (FIG.2B) confirmed the purity of the SUMO*-NS1 fusion protein. The correctconformation of the expressed and purified protein was confirmed bycommercial cross-serotype reactive Dengue Early NS1 capture ELISA(Panbio® Diagnostics, Brisbane, Australia). SUMO tag is cleavable fromthe target, DENV4 rNS1 using SUMO protease without leaving no extraneousresidues attached to the target protein. Despite this we used SUMO*-DENVrNS1 fusion protein for immunization of mice. Our strategy has anadvantage over immunization with cleaved DENV4 NS1 because it avoids thetime and effort required to cleave the SUMO* tag from DENV4 NS1. Inaddition, it reduces the loss of protein during cleavage andre-purification steps to remove the SUMO* tag.

Characteristics of DENV-4 Monoclonal Antibodies. A total of sixMAb-secreting hybridoma clones were isolated from SUMO*-DENV4 rNS1fusion protein primed splenocytes with Sp2/0 cells. A combination ofthree methods: western blot assay, iELISA and cell-based ELISA were usedto select hybridoma clones that could secret MAbs reactive to DENV NS1.Three MAbs designated hereafter as 3H7A9, 6D4B10, and 8A6F2 hadreactivity to DENV4 rNS1, but not to the SUMO* tag. Also, these threeMAbs did not cross-react with any of rNS1s from the other threeheterologous DENV serotypes and other related Flaviviruses (Yellow FeverVirus (YFV) and West Nile Virus (WNV)). The remaining three MAbs10H10B5, 10H8F7, and 4B6C10 were found to be specific to the SUMO* tag(Table 2, FIG. 3, FIG. 4 and FIG. 5). This result was expected since themice were immunized with SUMO*-DENV4 rNS1 fusion protein, and therelative size of the SUMO* tag, approximately 11.5 kDa. The light-chainand heavy-chain isotypes of the six MAbs, including those which areDENV4 serotype-specific, were IgG2b and K, respectively. CompetitionELISA results (data not shown) in combination with result fromsequencing the three complementarity determining regions (CDR1, CDR2,and CDR3) of MAbs (Supplement Figure) revealed that our three DENV4 NS1serotype-specific MAbs are distinct with different binding regions onthe DENV4 NS1.

Anti-DENV4 Monoclonal Antibodies were DENV4 Specific and Bind toMonomeric, Dimeric, and Hexameric DENV4 NS1 Isoforms. DENV4 serotypespecificity of MAbs (3H7A9, 6D4B10, and 8A6F2) was demonstrated in fixedcell-based ELISA and in iELISA using both cell culture supernatants fromDENV1-DENV4 infected Vero cells culture and rNS1 s of DENV1 (NativeAntigen Company, Oxfordshire, UK). (Table 2 and 3). Western blotanalysis of supernatants from the six clones after SDS-PAGE separationof SUMO*-DENV4 rNS1 protein, as well as unfused DENV4 rNS1s, revealedthat the binding affinity of the three MAbs (3H7A9, 6D4B10, and 8A6F2)was to the linear epitopes on both monomeric and dimeric DENV4 NS1isoforms (FIG. 3). Additionally, these MAbs exhibited reactivity tosupernatants from DENV4-infected Vero cell culture, suggesting that thelinear epitopes targeted by these three MAbs could also present onhexameric DENV4 NS1. Results from these three assays, including theWestern blot analysis, indicate that the MAbs are capable ofrecognizing/binding to all three DENV4 NS1 isoforms (monomeric, dimericand hexameric) (Table 2, Table 3, and FIG. 3).

MAbs Serotype-Specificity.

TABLE 2 Isotype, epitope, and iELISA results of six anti-DENV4monoclonal antibodies iELISA (reactivity MAbs to different recombinantproteins) SUMO*- NS1 rNS1 rNS1 rNS1 rNS1 rNS1 rNS1 fusion MAb IsotypeEpitope DENV1 DENV2 DENV3 DENV4 WNV YFV SUMO protein 10H8F7 IgG2b/KLinear − − − − − − + + 4B6C10 IgG2b/K Linear − − − − − − + + 3H7A9IgG2b/K Linear − − − + − − − + 8A6F2 IgG2b/K Linear − − − + − − − +10H10B5 IgG2b/K Linear − − − − − − + + 6D4B10 IgG2b/K Linear − − − + − −− + rNS1 = recombinant non-structural protein 1 DENV = dengue virusesSUMO = small-ubiquitin-like modifier YFV = Yellow Fever Virus WNV = WestNile Virus

TABLE 3 Monoclonal antibody reactivity to native DENV1-4 NS1 in cellbased ELISA and iELISA Reactivity to native NS1 Cell-Based ELISA (NS1 onsurface iELISA (NS1 in Vero cell culture infected Vero cells)supernatant) MAb DENV1 DENV2 DENV3 DENV4 DENV1 DENV2 DENV3 DENV4 10H8F7− − − − − − − − 4B6C10 − − − − − − − − 3H7A9 − − − + − − − + 8A6F2 − −− + − − − + 10H10B5 − − − − − − − − 6D4B10 − − − + − − − +

DENV NS1 Capture ELISA is Specific to DENV4. Results from competitionELISA (data not shown) confirmed that the three MAbs 3H7A9, 6D4B10, and8A6F2 recognized distinct epitopes on DENV4 NS1, indicating a pair ofthese MAbs may be able to be used to develop new NS1 (e.g., DENV4 NS1)specific detection tests/assays. However, Mab 3H7A9 had a lower relativeaffinity (FIG. 4) and was not utilized for development of the NS1capture assay of the present disclosure. Thus, only MAbs 6D4B10 and8A6F2 were utilized to develop the NS1 capture ELISA. Based on the limitof detection (LOD) curves, shown in FIG. 6, a matched pair8A6F2/biotinylated 6D4B10 had the lowest LOD while a matched pair6D4B10/B-8A6F2 was not robust to capture and/or detect DENV4 rNS1 evenat a higher concentration (FIG. 6B). Those matched MAbs pairs that didnot perform well could be due to, but in no way limited to: (i) thedenaturation/random orientation of 6D4B10 as a result of direct bindingto the microtiter plate, (ii) biotin molecules might have bound on theantigen binding region of 8A6F2, or (iii) a combination of (i) and (ii).Nevertheless, result from a direct ELISA (data not shown), in whichmicrotiter plate-wells were coated overnight with DENV4 rNS1, and boundantigen was detected by biotinylated 8A6F2, suggest that the loss ofantigen-antibody binding in an NS1 capture ELISA based on match pair6D4B10/biotinylated 8A6F2 was not linked to the biotinlyation.

Determination of the best working concentration of capture and detectionwas done by checkerboard titration and the optimal concentrations forcoating MAb 8A6F2 and detection MAb biotinylated 6D4B10 were found to be10 μg/mL and 1:2000 dilution, respectively.

After optimization of the NS1 capture ELISA specific to DENV4, theELISA's utility was established and validate using culture supernatantsobtained from DENV1-4 infected Vero cells. As expected, only culturesupernatants from DENV4-infected Vero cells showed reactivity whereasculture supernatants from the other three DENV serotypes-infected Verocell showed no reactivity.

Discussion of the Examples. DENV4 NS1 was expressed using SUMO fusiontechnology in Sf21 insect cells. The Examples here demonstrate that DENVNS1 protein SUMO* expression was superior to E. coli and standardbaculovirus antigen expression because SUMO* tagged DENV4 rNS1 wassecreted as a soluble molecule in culture medium. Furthermore, thepurified SUMO* tagged DENV NSA1 did not require a complex multi-stepprocess to solubilize and refold the expressed DENV NS1. To theinventor's knowledge, this is the first report regarding a successfulexpression of soluble and stable DENV4 NS1 with correct folding throughthe use of SUMO fusion technology. The present application demonstratesthe utility of purified SUMO*-DENV rNS1 for the generation of MAbs thatcould be used to develop immunodiagnostic tests for Dengue.

Production of a properly-folded soluble NS1 protein appears to becrucial for the development of MAbs, which are reactive to epitopes onhexameric NS1. Isolation and purification of proteins expressed in E.coli, require solubilization in harsh detergents (e.g., SDS and urea),which denatures the target protein and require complex refoldingprocesses. DENV NS1 expression in E. coli often resulted in insolubleaggregates (i.e., inclusion bodies). Even though it is possible toattain the correct 3-D configuration of the denatured protein followingrefolding, there is no guarantee this will occur. Expression of rNS1 inSf insect cell lines, such as Sf9 and SF21, using a baculovirusexpression system has been utilized, but in the inventor's experience(unpublished data), the expressed rNS1 protein also remained insoluble,and required solubilization and refolding.

The present disclosure provides six Mab-secreting hybridomas reactive tothe SUMO*-DENV4 NS1 fusion protein were generated, of which three werereactive to the SUMO* tag, thereby indicating that the tag is highlyimmunogenic. The remaining three MAbs (3H7A9, 6D4B10, and 8A6F2) werefound to be DENV4 serotype-specific.

Serotype-specific anti-DENV NS1 MAbs is crucial for the development ofNS1 test/assay that detects dengue virus infection early (e.g., early inthe course of Dengue illness) and simultaneously determines the infectedDENV serotype. The MAbs (3H7A9, 6D4B10 and 8A6F2) developed in thepresent disclosure are highly specific to DENV4, as demonstrated by twodifferent methods, cell-based ELISA, and iELISA. Of the threeserotype-specific MAbs developed, 6D4B10 and 8A6F2 show higher affinityto the hexameric DENV4 rNS1 and dimeric DENV4 NS1 expressed in Verocells. Additionally, the Western blot assay results under reducingconditions demonstrated that the reactivity to monomeric DENV rNS1 waswith lower affinity.

A competition ELISA was performed to determine if the three MAbs bind tothe same or distinct epitopes of DENV4 NS1. The results (data not shown)confirmed that all three MAbs (3H7A9, 6D4B10 and 8A6F2) bind to distinctnon-overlapping regions and can be used to develop sandwichimmunodiagnostic assays. The distinctness of these three MAbs wasfurther verified by sequencing the three complementary determiningregions (CDR1, CFR2, and CDR3) of the MAbs. MAb 3H7A9 was DENV4serotype-specific, but had weak affinity to DENV4, and was, therefore,not used for assay development. MAbs 6D4B10 and 8A6F2 were chosen todevelop a NS1 capture ELISA. In order to use biotin-streptavidin basedcapture ELISA format, different pairs of unbiotinylated and biotinylatedMAbs were evaluated. Subsequently, a match pair of unbiotinylated 8A6F2as a capture antibody and biotinylated (B)-6D4B10 as a detectionantibody provided the most robust results compared to 6D4B10 as thecapture antibody and (B)-8A6F2 as the detector antibody. The reason forthe poor performance of the later matched pair of MAbs may be due to:(i) the denaturation/random orientation of 6D4B10 as a result of directbinding to the microtiter plate, (ii) biotin molecules binding to theantigen binding region of 8A6F2, or (iii) a combination of (i) and (ii).Nevertheless, a direct ELISA (data not shown), in which microtiterplate-wells were coated with DENV4 rNS1 and detected by (B)-8A6F2),showed reactivity of the biotinylated 8A6F2 to DENV4 fNSA1 protein,suggesting the loss of antigen-antibody binding in the NS1 capture ELISAformat (captured with 6D4B10 and detected with (B)-8A6F2) was not due tothe biotinlyation. As such, this MAb pair (6D4B10/(B)-8A6F2) mayfunction efficiently if one avoids direct binding of 6D4B10 to thesurface of the microplate.

By using lower affinity capture Mab, 6D4B10 immobilization method or alinker, the DENV4 NS1 specific capture ELISA was developed using 8A6F2as a capture antibody and (B)-6D4B10 as detection antibody. The DENV4serotype-specific ELISA was assessed using rNS1 of Flaviviruses,including all four DENV serotypes and culture supernatants from Verocell-infected with all four DENV serotypes. The results confirmed thatthese MAbs specifically bound to DENV4 in the NS1 capture ELISA.

The inventors sought to develop a DENV4 ELISA because prior NS1 testsfor detecting DNV4 infections have limited sensitivity. Recent studiesusing retrospective samples from South America demonstrate lower DENV4sensitivity for seven commercially available NS1 Ag tests, as comparedto the other three serotypes. Also, a meta-analysis for DENV detectionin Asia also demonstrated that the lowest sensitivity of commercial NS1Ag tests is for the DENV4 detection. Furthermore, studies conducted inBrazil demonstrated the lower sensitivity of Platelia NS1 Ag ELISA(Bio-Rad®, Hercules, Calif., USA) for DENV4 detection. Collectively,these data suggest the need for development of new NS1 Ag detection testwith higher sensitivity to DENV4.

Another factor that could have contributed to the poor sensitivity ofNS1 Ag detection tests for DENV4 is the low level of expression of NS1in DENV4-infected patients, as compared to dengue patients infected withthe other three serotypes. An NS1 capture ELISA specific to DENV4 couldimprove the detection of DENV4 cases and a serotype-specific NS1 Ag testcan identify dengue and the infecting DENV serotype. NS1 ELISAs for eachone of the four DENV serotypes have been previously described. However,none of these ELISAs are commercially available. As a result, the onlycommonly used laboratory methods to determine DENV serotype currently isRT-PCR. There are two main advantages in identifying the DENV serotype:(i) risk factors for severe dengue have associated to more pathogenicDENV serotypes, and (ii) the sequence of infection of DENV serotype inprimary and secondary infections is also believed to be a risk factorfor severe disease. Taken together, there is a need for a DENV4serotype-specific NS1 tests that can detect NS1 in the serum ofDENV4-infected patients at the lowest possible LOD.

The three DENV4-specific MAbs directed against SUMO*-DENV4 rNS1 fusionprotein were generated. Based on characterization results andcompetition ELISA, the NS1 capture ELISA for potential early detectionDENV4 infection was established using a combination of two highlysensitive MAbs (8A6F2 as coating antibody and biotinylated 6D4B10 asdetection antibody) and an optimized protocol was developed. The assaywas sensitive and specific to DENV4 with no cross-reactivity to thethree other DENV serotypes and other heterologous Flaviviruses. TheExamples of the present disclosure indicate that the developed MAbs areuseful reagents for the development of immunodiagnostic assays (such as,ELISAs and lateral flow assays) that specifically detect DENV4infection. The DENV serotype-specific MAbs described herein have thepotential to change the way we detect dengue by providing tests that areuser friendly to resource poor regions to adopt them for diagnosingdengue. Given that the current commercial NS1 tests are less sensitivityto DENV4, the present test/assay could also provide an alternative testin regions where DENV4 circulates.

Specific Embodiments

According to an aspect, the present disclosure provides an antibody orantigen-binding fragment thereof that binds specifically to Dengue virusserotype 4 (DENV4), wherein the antibody or antigen-binding fragmentcomprises: a heavy chain variable region that comprises at least one CDRamino acid sequence selected from the group consisting of: SGYNWH,YIHYSGGTNYNPSLKS, RTGTVPFAY, SYVMH, YLNPYNDDTKYNEKFKG, and GPPYALDY.

In any aspect or embodiment described herein, the antigen-bindingfragment is selected from the group consisting of a Fab fragment, aF(ab)′, a F(ab)′2 fragment, or a single-chain variable fragments(scFvs).

In any aspect or embodiment described herein, the antibody orantigen-binding fragment thereof is specific for the DENV4Non-structural protein 1 (NS1).

In any aspect or embodiment described herein, the antibody orantigen-binding fragment thereof of further comprises a light chainvariable region that comprises at least one CDR amino acid sequenceselected from the group consisting of: SVSSSISSSNLH, GTSNLAS, QQWSSYPLT,RASQDISNYLN, YTSRLHS, and QQGNTLPRT.

In any aspect or embodiment described herein, the CDR amino acidsequence of the heavy chain variable region is selected from the groupconsisting of: SGYNWH, YIHYSGGTNYNPSLKS, RTGTVPFAY; and the CDR aminoacid sequence of the light chain variable region is selected from thegroup consisting of: SVSSSISSSNLH, GTSNLAS, and QQWSSYPLT.

In any aspect or embodiment described herein, the CDR amino acidsequence of the heavy chain variable region is selected from the groupconsisting of: SYVMH, YLNPYNDDTKYNEKFKG, and GPPYALDY; and the CDR aminoacid sequence of the light chain variable region is selected from thegroup consisting of: RASQDISNYLN, YTSRLHS, and QQGNTLPRT.

In any aspect or embodiment described herein, the heavy chain variableregion comprises the amino acid sequence of:

DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYNWHWIRQFPGNKLEWMGYIHYSGGTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARRT GTVPFAYWGQGTLVTVSA,or EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYLNPYNDDTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAYGP PYALDYWGQGTSVTVSS.

In any aspect or embodiment described herein, the light chain variableregion comprises the amino acid sequence of:

EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTSNLASGYPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFG GGTKLEIK, orDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVTLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPRTFGG GTKLEIK.

In any aspect or embodiment described herein, the heavy chain variableregion comprises the amino acid sequence of

DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYNWHWIRQFPGNKLEWMGYIHYSGGTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARRT GTVPFAYWGQGTLVTVSA;and the light chain variable region comprises the amino acid sequence of

EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFG GGTKLEIK.

In any aspect or embodiment described herein, the heavy chain variableregion comprises the amino acid sequence of

EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYLNPYNDDTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAYGP PYALDYWGQGTSVTVSS;and the light chain variable region comprises the amino acid sequence of

DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVTLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPRTFGG GTKLEIK.

In any aspect or embodiment described herein, the antibody is 3H7A9.

In any aspect or embodiment described herein, the antibody is 6D4B10.

In any aspect or embodiment described herein, the fragment of theantibody comprises an antigen-binding site/region of 3H7A9.

In any aspect or embodiment described herein, the fragment of theantibody comprises an antigen-binding site/region of 6D4B10.

According to another aspect, the present disclosure provides apharmaceutical composition comprising the antibody or the fragmentthereof of the present disclosure and a pharmaceutically acceptablecarrier.

According to a further aspect, the present disclosure provides a methodof diagnosing or detecting a DENV4 infection. The method comprises:contacting a sample from a patient with the antibody or antigen-bindingfragment thereof of the present disclosure; and detecting the binding ofNS1; and optionally administering an effective amount of a treatmenteffective for ameliorating at least one symptom of DENV4 infection.

In any aspect or embodiment described herein, contacting the bloodsample comprises: contacting the blood sample to an immobilized antibodyor antigen-binding fragment thereof of the present disclosure; andcontacting the antibody retained DENV4 virion with a second antibody orantigen-binding fragment thereof of the present disclosure.

In any aspect or embodiment described herein, the immobilized antibodyor antigen-binding fragment thereof and the second antibody or fragmentthereof are different antibodies.

In any aspect or embodiment described herein, the secondary antibody orantigen-binding fragment thereof is linked to (e.g., chemically orcovalent linked to) a detectable label.

In any aspect or embodiment described herein, the detectable label isselected from the group consisting of an enzyme, biotin, streptavidin, aradioactive molecule, and an immunofluorescent protein or dye.

In any aspect or embodiment described herein, when the detectable labelis biotin or streptavidin, the method further comprises contacting acomplex comprising a NS1 and the labeled antibody with a detectionmolecule that comprises streptavidin or biotin, respectively, linked toan immunofluorescent protein or dye or an enzyme.

In any aspect or embodiment described herein, the antibody is linked to(e.g., chemically or covalent linked to) a detectable label andoptionally a bead, particle, or nanoparticle.

In any aspect or embodiment described herein, the detectable label isselected from the group consisting of an enzyme, biotin, streptavidin, aradioactive molecule, and an immunofluorescent protein or dye.

In any aspect or embodiment described herein, when the detectable labelis biotin or streptavidin, the method further comprises contacting acomplex comprising a NS1 and the labeled antibody with a detectionmolecule that comprises streptavidin or biotin, respectively, linked toan immunofluorescent protein or dye.

In any aspect or embodiment described herein, the bead, particle, ornanoparticle is a magnetic bead.

In any aspect or embodiment described herein, wherein the method furthercomprises separating a complex comprising NS1 and the labeled antibodyor antigen-binding fragment thereof via the bead, particle ornanoparticle for detecting the binding of NS1.

In any aspect or embodiment described herein, the sample comprises ablood or a tissue sample.

In any aspect or embodiment described herein, the method furthercomprises administering at least one agent selected from the groupconsisting of an antibody or antigen-binding fragment of the presentdisclosure, the pharmaceutical composition of the present disclosure,acetaminophen, an analgesic with acetaminophen, and isotonic crystalloidsolution, wherein the agent is effective at ameliorating or treating atleast one system of the infection.

According to yet another aspect, the present disclosure provides amethod of treating a DENV4 infection in a subject. The method comprises:administering to a subject in the need thereof an effective amount ofthe antibody or antigen-binding fragment thereof, or the pharmaceuticalcomposition of the present disclosure, wherein the administering iseffective at treating the infection.

According to an additional aspect, the present disclosure provides amethod of producing/making a DENV NS1 specific antibody or fragmentthereof. The method comprises: providing a nucleic acid expressing DENVNS1 fusion protein with a solubility and stability tag; producing amultimeric DENV NS1 complex; and immunizing an animal with themultimeric DENV NS1 complex, wherein immunizing the animal produces theDENV NS1 specific antibody.

In any aspect or embodiment described herein, wherein the method furthercomprises preparing at least one hybridoma from spleen cells of theimmunized animal.

In any aspect or embodiment described herein, the solubility andstability tag includes a secretion signal.

In any aspect or embodiment described herein, the solubility andstability tag is a small ubiquitin-like modifier (SUMO) and/or thesecretion signal is gp67.

In any aspect or embodiment described herein, providing the nucleic acidexpressing DENV NS1 fusion protein comprises inserting the Dengue virusNS1 into a vector comprising the solubility and stability tag andoptionally, the secretion signal.

In any aspect or embodiment described herein, the DENV NS1 is DENV4 NS1.

In any aspect or embodiment described herein, producing the multimericDENV NS1 complex is performed with a eukaryotic expression system.

In any aspect or embodiment described herein, producing a multimericDENV NS1 complex includes a host cell comprising a vector that expressesa serotype specific DENV NS1 antibody.

In any aspect or embodiment described herein, the host cell is aeukaryotic cell.

In any aspect or embodiment described herein, the eukaryotic cell is aChinese hamster ovary (CHO) cell, a NS0 murine myeloma cell, PER.C6®human cells, an insect cell line, Sf9, or Sf21.

In any aspect or embodiment described herein, the eukaryotic expressionsystem is a baculovirus expression system or a vaccinia virus expressionsystem.

In any aspect or embodiment described herein, producing a multimericDENV NS1 complex comprises infecting eukaryotic cells with a baculavirusexpressing the DENV NS1 fusion protein.

In any aspect or embodiment described herein, the baculavirus expressingthe DENV NS1 fusion protein is prepared by at least one of: transforminga bacteria with a vector comprising the DENV NS1 fusion protein;selecting a vector-transformed bacteria; extracting/purifying the vectorfrom the vector-transformed bacteria; transforming a bacteria comprisinga baculovirus shuttle vector; selecting a bacteria with a recombinantDENV NS1 fusion protein-baculovirus vector; extracting/purifying therecombinant DENV NS1 fusion protein-baculovirus vector; transfecting aeukaryotic cell with the recombinant DENV NS1 fusion protein-baculovirusvector; or collecting cell culture supernatant comprising thebaculavirus expressing the DENV NS1 fusion protein.

In any aspect or embodiment described herein, immunizing the animal withthe multimeric DENV NS1 complex includes at least one of: administeringthe multimeric DENV NS1 complex to the animal at least two times;isolating at least one primed spleen cell from the animals; fusing theprimed spleen cell with a myeloma cell; or selecting a hybridoma cellexpressing the DENV NS1 specific antibody.

In any aspect or embodiment described herein, the method furthercomprises humanizing the DENV NS1 specific antibody.

In any aspect or embodiment described herein, the method furthercomprises treating the DENV NS1 specific antibody to produce anantigen-binding fragment thereof.

In any aspect or embodiment described herein, treating comprisescontacting the DENV NS1 specific antibody with an agent selected fromthe group consisting of (i) pepsin, (ii) papain, and (iii) pepsin andβ-mercaptoethanol.

According to yet an additional aspect, the present disclosure providesan antibody or antigen-binding fragment thereof that binds specificallyto Dengue virus serotype 4 (DENV4), wherein the antibody is: 8A6F2 orthe antigen-binding fragment comprises at least at least one heavy chainor light chain CDR amino acid sequence from an antigen-bindingsite/region of 8A6F2; 3H7A9 or the antigen-binding fragment comprises atleast at least one heavy chain or light chain CDR amino acid sequencefrom an antigen-binding site/region of 3H7A9; or 6D4B10 or theantigen-binding fragment comprises at least at least one heavy chain orlight chain CDR amino acid sequence from an antigen-binding site/regionof 6D4B10.

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables, andappendices, as well as patents, applications, and publications, referredto above, are hereby incorporated by reference.

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1. An antibody or antigen-binding fragment thereof that bindsspecifically to Dengue virus serotype 4 (DENV4), wherein the antibody orantigen-binding fragment comprises: a heavy chain variable region thatcomprises at least one CDR amino acid sequence selected from the groupconsisting of: SGYNWH, YIHYSGGTNYNPSLKS, RTGTVPFAY, SYVMH,YLNPYNDDTKYNEKFKG, and GPPYALDY.
 2. The antibody or antigen-bindingfragment thereof according to claim 1, wherein the antigen-bindingfragment is selected from the group consisting of a Fab fragment, aF(ab)′, a F(ab)′2 fragment, or a single-chain variable fragments(scFvs).
 3. The antibody or antigen-binding fragment thereof accordingto claim 1, wherein the antibody or antigen-binding fragment thereof isspecific for the DENV4 Non-structural protein 1 (NS1).
 4. The antibodyor antigen-binding fragment thereof according to claim 1, furthercomprising a light chain variable region that comprises at least one CDRamino acid sequence selected from the group consisting of: SVSSSISSSNLH,GTSNLAS, QQWSSYPLT, RASQDISNYLN, YTSRLHS, and QQGNTLPRT.
 5. The antibodyor antigen-binding fragment thereof according to claim 1, wherein: theCDR amino acid sequence of the heavy chain variable region is selectedfrom the group consisting of: SGYNWH, YIHYSGGTNYNPSLKS, RTGTVPFAY; andthe CDR amino acid sequence of the light chain variable region isselected from the group consisting of: SVSSSISSSNLH, GTSNLAS, andQQWSSYPLT.
 6. The antibody or antigen-binding fragment thereof accordingto claim 1, wherein: the CDR amino acid sequence of the heavy chainvariable region is selected from the group consisting of: SYVMH,YLNPYNDDTKYNEKFKG, and GPPYALDY; and the CDR amino acid sequence of thelight chain variable region is selected from the group consisting of:RASQDISNYLN, YTSRLHS, and QQGNTLPRT.
 7. The antibody or antigen-bindingfragment thereof according to claim 1, wherein the heavy chain variableregion comprises the amino acid sequence of:DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYNWHWIRQFPGNKLEWMGYIHYSGGTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARRT GTVPFAYWGQGTLVTVSA,or EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYLNPYNDDTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAYGP PYALDYWGQGTSVTVSS.


8. The antibody or antigen-binding fragment thereof according to claim1, wherein the light chain variable region comprises the amino acidsequence of: EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFG GGTKLEIK, orDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVTLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPRTFGG GTKLEIK.


9. The antibody or antigen-binding fragment thereof according to claim1, wherein: the heavy chain variable region comprises the amino acidsequence of DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYNWHWIRQFPGNKLEWMGYIHYSGGTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARRT GTVPFAYWGQGTLVTVSA;

and the light chain variable region comprises the amino acid sequence ofEIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFG GGTKLEIK.


10. The antibody or antigen-binding fragment thereof according to claim1, wherein: the heavy chain variable region comprises the amino acidsequence of EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYLNPYNDDTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAYGP PYALDYWGQGTSVTVSS;

and the light chain variable region comprises the amino acid sequence ofDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVTLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPRTFGG GTKLEIK.


11. The antibody or antigen-binding fragment thereof according to claim1, wherein the antibody is 3H7A9.
 12. The antibody or antigen-bindingfragment thereof according to claim 1, wherein the antibody is 6D4B10.13. The antibody or antigen-binding fragment thereof according to claim1, wherein the fragment of the antibody comprises an antigen-bindingsite/region of 3H7A9.
 14. The antibody or antigen-binding fragmentthereof according to claim 1, wherein the fragment of the antibodycomprises an antigen-binding site/region of 6D4B10.
 15. A pharmaceuticalcomposition comprising the antibody or the fragment thereof of claim 1,and a pharmaceutically acceptable carrier.
 16. A method of diagnosing ordetecting a DENV4 infection, the method comprising: contacting a samplefrom a patient with the antibody or antigen-binding fragment thereofaccording to claim 1; and detecting the binding of NS1; and optionallyadministering an effective amount of a treatment effective forameliorating at least one symptom of DENV4 infection.
 17. The methodaccording to claim 16, wherein contacting the blood sample comprises:contacting the blood sample to an immobilized antibody orantigen-binding fragment thereof according to claim 1; and contactingthe antibody retained DENV4 virion with a second antibody orantigen-binding fragment thereof according to claim
 1. 18. The methodaccording to claim 16, wherein the immobilized antibody orantigen-binding fragment thereof and the second antibody or fragmentthereof are different antibodies.
 19. The method according to claim 18,wherein the secondary antibody or antigen-binding fragment thereof islinked to a detectable label.
 20. The method according to claim 19,where the detectable label is selected from the group consisting of anenzyme, biotin, streptavidin, a radioactive molecule, and animmunofluorescent protein or dye.
 21. The method according to claim 20,wherein when the detectable label is biotin or streptavidin, the methodfurther comprises contacting a complex comprising a NS1 and the labeledantibody with a detection molecule that comprises streptavidin orbiotin, respectively, linked to an immunofluorescent protein or dye oran enzyme.
 22. The method according to claim 16, wherein the antibody islinked to a detectable label and optionally a bead, particle, ornanoparticle.
 23. The method according to claim 22, wherein thedetectable label is selected from the group consisting of an enzyme,biotin, streptavidin, a radioactive molecule, and an immunofluorescentprotein or dye.
 24. The method according to claim 23, wherein when thedetectable label is biotin or streptavidin, the method further comprisescontacting a complex comprising a NS1 and the labeled antibody with adetection molecule that comprises streptavidin or biotin, respectively,linked to an immunofluorescent protein or dye.
 25. The method accordingto claim 22, wherein the bead, particle, or nanoparticle is a magneticbead.
 26. The method according to claim 22, further comprisingseparating a complex comprising NS1 and the labeled antibody orantigen-binding fragment thereof via the bead, particle or nanoparticlefor detecting the binding of NS1.
 27. The method according to claim 16,wherein the sample comprises a blood or a tissue sample.
 28. The methodaccording to claim 16, further comprising administering at least oneagent selected from the group consisting of the antibody according toclaim 1, a pharmaceutical composition comprising the antibody or thefragment thereof of claim 1, acetaminophen, an analgesic withacetaminophen, and isotonic crystalloid solution, wherein the agent iseffective at ameliorating or treating at least one system of theinfection.
 29. A method of treating a DENV4 infection in a subject, themethod comprising: administering to a subject in the need thereof aneffective amount of the antibody according to claim 1, or apharmaceutical composition comprising the antibody or the fragmentthereof of claim 1, wherein administering is effective at treating orameliorating at least one symptom the infection.
 30. A method ofproducing/making a DENV NS1 specific antibody or fragment thereof, themethod comprising: providing a nucleic acid expressing a DENV NS1 fusionprotein with a solubility and stability tag; producing a multimeric DENVNS1 complex; and immunizing an animal with the multimeric DENV NS1complex, wherein immunizing the animal produces the DENV NS1 specificantibody.
 31. The method of claim 30, further comprising preparing atleast one hybridoma from spleen cells of the immunized animal.
 32. Themethod of claim 30, wherein the solubility and stability tag includes asecretion signal.
 33. The method of claim 30, wherein at least one ofthe solubility and stability tag is a small ubiquitin-like modifier(SUMO), the secretion signal is gp67, or both.
 34. The method of claim30, wherein providing the nucleic acid expressing DENV NS1 fusionprotein comprises inserting the Dengue virus NS1 into a vectorcomprising the solubility and stability tag and optionally, thesecretion signal.
 35. The method of claim 30, wherein the DENV NS1 isDENV4 NS1.
 36. The method of claim 30, wherein producing the multimericDENV NS1 complex is performed with a eukaryotic expression system. 37.The method of claim 30, wherein producing a multimeric DENV NS1 complexincludes a host cell comprising a vector that expresses a serotypespecific DENV NS1 antibody.
 38. The method of claim 37, wherein the hostcell is a eukaryotic cell.
 39. The method of claim 38, wherein theeukaryotic cell is a Chinese hamster ovary (CHO) cell, a NS0 murinemyeloma cell, PER.C6® human cells.
 40. The method of claim 36, whereinthe eukaryotic expression system is a baculovirus expression system or avaccinia virus expression system.
 41. The method of claim 30, whereinproducing a multimeric DENV NS1 complex comprises infecting eukaryoticcells with a baculovirus expressing the DENV NS1 fusion protein.
 42. Themethod of claim 30, wherein the baculovirus expressing the DENV NS1fusion protein is prepared by at least one of: transforming a bacteriawith a vector comprising the DENV NS1 fusion protein; selecting avector-transformed bacteria; extracting/purifying the vector from thevector-transformed bacteria; transforming a bacteria comprising abaculovirus shuttle vector; selecting a bacteria with a recombinant DENVNS1 fusion protein-baculovirus vector; extracting/purifying therecombinant DENV NS1 fusion protein-baculovirus vector; transfecting aeukaryotic cell with the recombinant DENV NS1 fusion protein-baculovirusvector; collecting cell culture supernatant comprising the baculovirusexpressing the DENV NS1 fusion protein; or a combination thereof. 43.The method of claim 30, wherein immunizing the animal with themultimeric DENV NS1 complex includes at least one of: administering themultimeric DENV NS1 complex to the animal at least two times; isolatingat least one primed spleen cell from the animals; fusing the primedspleen cell with a myeloma cell; selecting a hybridoma cell expressingthe DENV NS1 specific antibody; or a combination thereof.
 44. The methodof claim 30, further comprising humanizing the DENV NS1 specificantibody.
 45. The method of claim 30, further comprising treating theDENV NS1 specific antibody to produce an antigen-binding fragmentthereof.
 46. The method of claim 45, wherein treating comprisescontacting the DENV NS1 specific antibody with an agent selected fromthe group consisting of (i) pepsin, (ii) papain, and (iii) pepsin andβ-mercaptoethanol.